MN/CA IX and EGFR Pathway Inhibition

ABSTRACT

The invention is based upon the discovery that the EGFR pathway can stimulate a previously unknown tumorigenic function of CA IX, via phosphorylation of the sole tyrosine residue present in CA IX&#39;s intracellular domain. EGFR-phosphorylated CA IX then interacts with the p85 subunit of PI3K to activate Akt, which in turn is associated with anti-apototic function and increased cell survival. The latter finding indicates that there is a positive feedback loop for CA9 expression mediated by the PI3K pathway in preneoplastic/neoplastic diseases. Disclosed herein are novel therapeutic methods for treating preneoplastic/neoplastic diseases associated with abnormal MN/CA IX expression, using EGFR pathway inhibitors. Preferably, the EGFR pathway inhibitors are tyrosine kinase inhibitors or EGFR-specific antibodies. Further disclosed are methods for patient therapy selection for EGFR pathway inhibitors, preferably in combination with other cancer therapies, based on detection of abnormal MN/CA9 gene expression in preneoplastic/neoplastic tissues.

FIELD OF THE INVENTION

The present invention is in the general area of medical genetics and inthe fields of biochemical engineering, immunochemistry and oncology.More specifically, it relates to the MN gene—a cellular gene consideredto be an oncogene, known alternatively as MN/CA9, CA9, or carbonicanhydrase 9, which gene encodes the oncoprotein now known alternativelyas the MN protein, the MN/CA IX isoenzyme, MN/CA IX, carbonic anhydraseIX, CA IX, the MN/G250 or the G250 protein.

More specifically, the instant invention is based upon the discovery ofa potential tumorigenic role of MN/CA IX's intracellular domain (IC):the sole tyrosine moiety of MN/CA IX present in its IC domain can bephosphorylated in an EGFR-dependent manner and leads to activation ofAkt. As the Akt pathway has a distinct antiapoptotic function, thatdiscovery has important applications for the therapy ofpreneoplastic/neoplastic diseases characterized by abnormal MN/CA9 geneexpression, and for making clinical decisions on cancer treatment.

BACKGROUND OF THE INVENTION

As indicated above, the MN gene and protein are known by a number ofalternative names, which names are used herein interchangeably. The MNprotein was found to bind zinc and have carbonic anhydrase (CA) activityand is now considered to be the ninth carbonic anhydrase isoenzyme—MN/CAIX or CA IX [Opaysky et al., Genomics, 33: 480-487 (1996)]. According tothe carbonic anhydrase nomenclature, human CA isoenzymes are written incapital roman letters and numbers, whereas their genes are written initalic letters and arabic numbers. Alternatively, “MN” is used herein torefer either to carbonic anhydrase isoenzyme IX (CA IX)proteins/polypeptides, or carbonic anhydrase isoenzyme 9 (CA9) gene,nucleic acids, cDNA, mRNA etc. as indicated by the context.

The MN protein has also been identified with the G250 antigen. Uemura etal. [J. Urol. 157 (4 Suppl.): 377 (Abstract 1475; 1997)] states:“Sequence analysis and database searching revealed that G250 antigen isidentical to MN, a human tumor-associated antigen identified in cervicalcarcinoma (Pastorek et al., 1994).”

Zavada et al., International Publication No. WO 93/18152 (published Sep.16, 1993) and U.S. Pat. No. 5,387,676 (issued Feb. 7, 1995) describe thediscovery of the MN gene and protein. The MN gene was found to bepresent in the chromosomal DNA of all vertebrates tested, and itsexpression to be strongly correlated with tumorigenicity. In general,oncogenesis may be signified by the abnormal expression of MN/CA IXprotein. For example, oncogenesis may be signified: (1) when MN/CA IXprotein is present in a tissue which normally does not express MN/CA IXprotein to any significant degree; (2) when MN/CA IX protein is absentfrom a tissue that normally expresses it; (3) when CA9 gene expressionis at a significantly increased level, or at a significantly reducedlevel from that normally expressed in a tissue; or (4) when MN/CA IXprotein is expressed in an abnormal location within a cell. WO 93/18152further discloses, among other MN-related inventions, MN/CA IX-specificmonoclonal antibodies (MAbs), including the M75 MAb and the VU-M75hybridoma that secretes the M75 MAb. The M75 MAb specifically binds toimmunodominant epitopes on the proteoglycan (PG) domain of the MN/CA IXproteins.

Zavada et al., International Publication No. WO 95/34650 (published Dec.21, 1995) provides in FIG. 1 the nucleotide sequences for a full-lengthMN cDNA [also provided herein in FIG. 6 (SEQ ID NO: 1)] clone isolatedas described therein, and the amino acid sequence [also provided hereinin FIG. 6 (SEQ ID NO: 2)] encoded by that MN cDNA. WO 95/34650 alsoprovides in FIG. 6 the nucleotide sequence for the MN promoter [SEQ IDNO: 24]. Those MN cDNA, promoter and amino acid sequences areincorporated by reference herein.

Zavada et al., International Publication No. WO 03/100029 (publishedDec. 4, 2003) discloses among other MN-related inventions, MN/CAIX-specific MAbs that are directed to non-immunodominant epitopes,including those on the carbonic anhydrase (CA) domain of the MN/CA IXprotein. An example of such a MN/CA IX-specific MAb is the V/10 MAb,secreted from the V/10-VU hybridoma.

The MN protein is now considered to be the first tumor-associatedcarbonic anhydrase isoenzyme that has been described. The carbonicanhydrase family (CA) includes eleven catalytically active zincmetalloenzymes involved in the reversible hydration-dehydration ofcarbon dioxide: CO₂+H₂0

HCO₃ ⁻+H⁺. CAs are widely distributed in different living organisms. TheCAs participate in a variety of physiological and biological processesand show remarkable diversity in tissue distribution, subcellularlocalization, and biological functions [Parkkila and Parkkila, Scand J.Gastroenterol., 31: 305-317 (1996); Potter and Harris, Br J Cancer, 89:2-7 (2003); Wingo et al., Biochem Biophys Res Commun, 288: 666-669(2001)]. Carbonic anhydrase IX, CA IX, is one of the most recentlyidentified isoenzymes [Opaysky et al., Genomics, 33: 480-487 (1996);Pastorek et al., Oncogene, 9: 2877-2888 (1994)]. Because of the CA IXoverexpression in transformed cell lines and in several humanmalignancies, it has been recognized as a tumor-associated antigen andlinked to the development of human cancers [Zavada et al., Int. J.Cancer, 54: 268-274 (1993); Liao et al., Am. J. Pathol., 145: 598-609(1994); Saarnio et al., Am J Pathol, 153: 279-285 (1998)].

MN/CA IX is a glycosylated transmembrane CA isoform with a uniqueN-terminal proteoglycan-like extension. Through transfection studies ithas been demonstrated that MN/CA IX can induce the transformation of 3T3cells [Opaysky et al., Genomics, 33: 480-487 (1996); Pastorek et al.,Oncogene, 9: 2877-2888 (1994)].

The MN protein was first identified in HeLa cells, derived from a humancarcinoma of cervix uteri. Many studies, using the MN-specificmonoclonal antibody (MAb) M75, have confirmed the diagnostic/prognosticutility of MN in diagnosing/prognosing precancerous and cancerouscervical lesions [Liao et al., Am. J. Pathol., 145: 598-609 (1994); Liaoand Stanbridge, Cancer Epidemiology, Biomarkers & Prevention, 5: 549-557(1996); Brewer et al., Gynecologic Oncology 63: 337-344 (1996)].Immunohistochemical studies with the M75 MAb of cervical carcinomas anda PCR-based (RT-PCR) survey of renal cell carcinomas have identified MNexpression as closely associated with those cancers and confirm MN'sutility as a tumor biomarker [Liao et al., Am. J. Pathol., 145: 598-609(1994); Liao and Stanbridge, Cancer Epidemiology, Biomarkers &Prevention, 5: 549-557 (1996); McKiernan et al., Cancer Res. 57:2362-2365 (1997)]. In various cancers (notably uterine cervical,ovarian, endometrial, renal, bladder, breast, colorectal, lung,esophageal, head and neck and prostate cancers, among others), MN/CA IXexpression is increased and has been correlated with microvessel densityand the levels of hypoxia in some tumors [Koukourakis et al., ClinCancer Res, 7: 3399-3403 (2001); Giatromanolaki et al., Cancer Res, 61:7992-7998 (2001)].

In tissues that normally do not express MN protein, MN/CA IX positivityis considered to be diagnostic for preneoplastic/neoplastic diseases,such as, lung, breast and cervical precancers/cancers [Swinson et al., JClin Oncol, 21: 473-482 (2003); Chia et al., J Clin Oncol, 19: 3660-3668(2001); Loncaster et al., Cancer Res, 61: 6394-6399 (2001)], among otherprecancers/cancers. Very few normal tissues have been found to expressMN protein to any significant degree. Those MN-expressing normal tissuesinclude the human gastric mucosa and gallbladder epithelium, and someother normal tissues of the alimentary tract. Paradoxically, MN geneexpression has been found to be lost or reduced in carcinomas and otherpreneoplastic/neoplastic diseases in some tissues that normally expressMN, e.g., gastric mucosa.

MN Regulation Under Hypoxia and Normoxia

Strong association of MN/CA IX with a broad range of tumors isprincipally related to its transcriptional regulation by hypoxia andhigh cell density, which appear to activate the MN/CA9 promoter throughtwo different, but interconnected pathways [Wykoff et al., Cancer Res.,60: 7075-7083 (2000); Lieskovska, et al., Neoplasma, 46: 17-24 (1999);Kaluz et al., Cancer Res., 62: 4469-4477 (2002)]. Those two pathways areactivated via stabilization of HIF-1α by hypoxia, and direct stimulationof MN/CA IX protein expression by the phosphotidylinositol-3-kinase(PI3K) pathway, respectively.

Hypoxia is a reduction in the normal level of tissue oxygen tension. Itoccurs during acute and chronic vascular disease, pulmonary disease andcancer, and produces cell death if prolonged. Pathways that areregulated by hypoxia include angiogenesis, glycolysis, growth-factorsignaling, immortalization, genetic instability, tissue invasion andmetastasis, apoptosis and pH regulation [Harris, A. L., Nature Reviews,2: 38-47 (January 2002)].

The central mediator of transcriptional up-regulation of a number ofgenes during hypoxia is the transcription factor. HIF-1 is composed oftwo subunits: a constitutively expressed HIF-1β and a rate-limitingHIF-1α, which is regulated by the availability of oxygen. Under hypoxia,HIF-1α skips modification of its conserved proline and asparagineresidues by oxygen-sensitive hydroxylases, thus avoiding degradationmediated by pVHL and inactivation mediated by FIH-1 (factor inhibitingHIF-1) [Maxwell et al., Nature, 399: 271-275 (1999); Jaakkola et al.,Science, 292: 468-472 (2001); Ivan et al., Science, 292: 464-468, 2001;Jaakkola, et al., Science, 292: 468-472 (2001); Mahon, et al., GenesDev., 15: 2675-2686 (2001)]. This leads to HIF-1α accumulation,dimerization with HIF-1β, binding to hypoxia response element (HRE)sites in the target genes, interaction with the cofactors andstimulation of the HIF-1 trans-activation capacity.

In the absence of oxygen, HIF-1 binds to HIF-binding sites within HREsof oxygen-regulated genes, thereby activating the expression of numeroushypoxia-response genes, such as erythropoietin (EPO), and theproangiogenic growth factor vascular endothelial growth factor (VEGF).In addition, HIF-1α can be up-regulated under normoxic conditions bydifferent extracellular signals and oncogenic changes transmitted viathe PI3K and MAPK pathways [Semenza, Biochem. Pharmacol., 64: 993-998(2002); Bardos and Ashcroft, BioEssays, 26: 262-269 (2004)]. WhereasPI3K activation results in an increased level of HIF-1α protein, MAPKactivation improves its trans-activation properties [Laughner, et al.,Mol. Cell. Biol., 21: 3995-4004 (2001); Richard et al., J. Biol. Chem.,274: 32631-32637 (1999)].

MN/CA IX was shown to be one of the most strongly hypoxia-inducibleproteins, via the HIF-1 protein binding to the hypoxia-responsiveelement of the MN promoter [Wykoff et al., Cancer Res, 60: 7075-7083(2000); Svastova et al., Exp Cell Res, 290: 332-345 (2003)]. Like otherHIF-1-regulated genes, the transcription of the MN gene is negativelyregulated by wild-type von Hippel-Lindau tumor suppressor gene [Ivanovet al., Proc Natl Acad Sci (USA), 95: 12596-12601 (1998)]. Low levels ofoxygen lead to stabilization of HIF-1α, which in turn leads to theincreased expression of MN [Wykoff et al., Cancer Res, 60: 7075-7083(2000)]. Areas of high expression of MN in cancers are linked to tumorhypoxia as reported in many cancers, and incubation of tumor cells underhypoxic conditions leads to the induction of MN expression [Wykoff etal., Cancer Res, 60: 7075-7083 (2000); Koukourakis et al., Clin CancerRes, 7: 3399-3403 (2001); Giatromanolaki et al., Cancer Res, 61:7992-7998 (2001); Swinson et al., J Clin Oncol, 21: 473-482 (2003); Chiaet al., J Clin Oncol, 19: 3660-3668 (2001); Loncaster et al., CancerRes, 61: 6394-6399 (2001)].

Key elements of the MN/CA9 promoter are the HIF-1 and SP1 bindingregions [Kaluz et al., Cancer Res. 63: 917-922 (2003)] [PR1-HREelement]. The MN/CA9 promoter sequence (−3/−10) between thetranscription start and PR1 contains a HRE element recognized by ahypoxia inducible factor 1 (HIF-1), which governs transcriptionalresponses to hypoxia [Wykoff et al., Cancer Res. 60: 7075-7083 (2000)].The promoter of the CA9 gene contains five regions protected in DNase Ifootprinting (PR1-PR5, numbered from the transcription start) [Kaluz etal., J. Biol. Chem., 274: 32588-32595 (1999)]. PR1 and PR2 bind SP1/3and AP1 transcription factors and are critical for the basic activationof CA9 transcription [Kaluz et al., J. Biol. Chem., 274: 32588-32595(1999); Kaluzova et al., Biochem. J., 359: 669-677 (2001)]. HIF-1strongly induces transcription of the CA9 gene in hypoxia, but for fullinduction requires a contribution of the SP1/3 transcription factorbinding to PR1 [Wykoff et al., Cancer Res. 60: 7075-7083 (2000); Kaluz,et al., Cancer Res., 63: 917-922 (2003)].

Regulation under normoxia also requires SP1 [Kaluz et al., Cancer Res.,62: 4469-4477 (2002)]. Upregulation of CA9 transcription in increasedcell density involves a mild pericellular hypoxia, depends uponcooperation of SP1 with HIF-1 at subhypoxic level and operates via thePI3K pathway [Kaluz et al., Cancer Res., 62: 4469-4477 (2002)]. Hypoxiaand cell density act in an additive fashion so that the highestexpression of CA9 is achieved under conditions of low oxygen at highdensity [Kaluz et al., Cancer Res., 62: 4469-4477 (2002)].

MN's Intracellular Region and the EGFR Pathway

As indicated above, CA9 expression is upregulated by both HIF-1α- andPI3K-dependent pathways, and both the PG and CA extracellular domains ofCA IX have predicted roles in tumorigenesis based on cell adhesion andcarbonic anhydrase activities. The invention disclosed herein is basedon the discovery of a potential tumorigenic role of a third CA IXdomain, the intracellular domain (IC). The inventor discloses finding CAIX to be associated with EGFR in lipid rafts in RCC cell lines, and thatthe sole tyrosine moiety of CA IX present in its IC domain can bephosphorylated in an EGFR-dependent manner. The inventor found evidencethat tyrosine-phosphorylated CA IX interacts with the regulatory subunitof PI3K (p85), resulting in activation of Akt. That finding indicatesthat there is a positive feedback loop for CA9 expression in RCC,mediated by the PI3K pathway, which may contribute to the aggressivenessof RCC. Based on those novel findings, the instant invention disclosestherapeutic methods targeted to the EGFR pathway which can be usedalone, or in combination with other MN-targeted methods, to treatpreneoplastic/neoplastic diseases characterized by abnormal MNexpression.

SUMMARY OF THE INVENTION

The subject invention is based upon the discovery of a potentialtumorigenic role of the intracellular domain (IC) of CA IX: the soletyrosine moiety present in the IC can be phosphorylated by the EGFRpathway, leading to CA IX interaction with the p85 regulatory subunit ofPI3K and resulting in activation of Akt. As Akt activation hasantiapoptotic effects, inhibiting phosphorylation of CA IX'sintracellular domain is considered to have important consequences fortumor biology.

The subject invention concerns the identification of a site within theCA IX intracellular domain [SEQ ID NO: 7] comprising a tyrosine residuewhich can be phosphorylated in an EGFR-dependent manner. EGFR pathwayinhibitors are then a new therapy for targeting tumors associated withabnormal CA9 expression, usually increased CA9 expression. Said EGFRpathway inhibitors may be targeted to any components of the EGFRpathway, including, for example, Ras, Raf, MEK, and ERK. Preferably,said EGFR pathway inhibitors are used in combination with otherMN-targeted therapies, such as CA IX-specific antibodies, CA IX-specificcarbonic anhydrase inhibitors, CA9 antisense therapies and/orPI3K-targeted therapies.

In one aspect, the instant invention is directed to a method of treatinga mammal, preferably a human, for a preneoplastic/neoplastic disease,wherein said disease is characterized by abnormal MN/CA9 geneexpression, comprising administering to said mammal a therapeuticallyeffective amount of a composition comprising an EGFR pathway inhibitor.Preferably, said EGFR pathway inhibitor is an EGFR tyrosine kinaseinhibitor or an anti-EGFR antibody. Preferably, said EGFR tyrosinekinase inhibitor is selected from gefitinib, erlotinib, lapatinib,canertinib and EKB-569, and said anti-EGFR antibody is selected fromcetuximab, panitumumab, nimotuzumab, matuzumab, and MDX-447. Said EGFRpathway inhibitor may be administered in an unmodified form, or may beconjugated to an antibody or biologically active antibody fragment whichspecifically binds MN/CA IX. In one preferred embodiment of theinvention, said EGFR pathway inhibitor is a bispecific antibody orantibody fragment having a specificity for EGFR and a specificity forMN/CA IX.

Preferably, said therapeutic methods further comprise administering tosaid mammal radiation and/or a therapeutically effective amount in aphysiologically acceptable formulation of one or more of the followingcompounds selected from the group consisting of: conventional anticancerdrugs, chemotherapeutic agents, different inhibitors of cancer-relatedpathways, bioreductive drugs, gene therapy vectors, CA9 antisenseoligonucleotides and vectors, CA IX-specific inhibitors, CA IX-specificantibodies and CA IX-specific antibody fragments that are biologicallyactive. Preferably, said inhibitors of cancer-related pathways areselected from HIF-1 α targeted therapies, VEGF-R targeted therapies,IL-2 and interferon-α, inhibitors of the MAPK pathway, inhibitors of thePI3K pathway; and/or said gene therapy vectors are targeted to hypoxictumors. Preferably, said inhibitor of the MAPK pathway is the bisaryl-urea Sorafenib (BAY 43-9006) or an omega-carboxypyridyl substitutedurea. Most preferably, said inhibitor of the MAPK pathway is the bisaryl-urea Sorafenib (BAY 43-9006).

Said preneoplastic/neoplastic disease characterized by abnormal MN/CA9gene expression can be that of many different tissues, for example,uterine, cervical, ovarian, endometrial, renal, bladder, breast,colorectal, lung, esophageal, and prostate, among many other tissues. Ofparticular interest are preneoplastic/neoplastic diseases of the breast,colon, rectum and of the urinary tract, as of the kidney, bladder andurethra. Renal cell carcinoma (RCC), and metastatic breast cancer arejust a couple of representative disease characterized by abnormally highlevels of MN/CA9 expression. Also, representative are mesodermal tumors,such as neuroblastomas and retinoblastomas; sarcomas, such asosteosarcomas and Ewing's sarcoma; melanomas; and gynecologicpreneoplastic/neoplastic diseases, particularly, of the uterine cervix,endometrium and ovaries, more particularly, cervical squamous cell,adrenosquamous, and glandular preneoplastic/neoplastic diseases,including adenocarcinoma, cervical metaplasia, and condylomas.

Exemplary preneoplastic/neoplastic diseases characterized by abnormalMN/CA9 gene expression are selected from the group consisting ofmammary, urinary tract, bladder, kidney, ovarian, uterine, cervical,endometrial, squamous cell, adenosquamous cell, vaginal, vulval,prostate, liver, lung, skin, thyroid, pancreatic, testicular, brain,head and neck, mesodermal, sarcomal, stomach, spleen, gastrointestinal,esophageal, and colon preneoplastic/neoplastic diseases. Preferably,said preneoplastic/neoplastic disease characterized by abnormal MN/CA9gene expression is kidney cancer, most preferably, renal cell carcinoma.Said disease may be either a normoxic or a hypoxic tumor.

In a second aspect, the invention concerns a method of therapy selectionfor a human patient with a preneoplastic/neoplastic disease, comprisingdetecting and quantifying the level of MN/CA9 gene expression in asample taken from the patient; and deciding to use EGFR pathway-directedtherapy to treat the patient based upon abnormal levels of MN/CA9 geneexpression in the patient's sample, usually based upon increased levelsof MN/CA9 expression above normal MN/CA9 expression levels. Preferably,said EGFR pathway-directed therapy comprises the use of a tyrosinekinase inhibitor or an anti-EGFR antibody. Said EGFR pathway inhibitormay be administered in an unmodified form, or may be conjugated to anantibody or biologically active antibody fragment which specificallybinds MN/CA IX. Said therapeutic methods may further compriseadministering to said human one or more additional therapies;preferably, said additional therapies target MN/CA9 expression or MN/CAIX enzymatic activity. Said additional therapies may comprise the use ofthe bis aryl-urea Sorafenib (BAY 43-9006) or an omega-carboxypyridylsubstituted urea. Most preferably, said additional therapy is the bisaryl-urea Sorafenib (BAY 43-9006).

Said preneoplastic/neoplastic sample would preferably be a tissue, cellor body fluid sample. A tissue sample could be, for example, aformalin-fixed, paraffin-embedded tissue sample or a frozen tissuesample, among other tissue samples. A body fluid sample could be, forexample, a blood, serum, plasma or urine sample, among other body fluidsamples.

Preferably, said detecting and quantifying step comprisesimmunologically detecting and quantifying the level of MN/CA IX proteinin said sample, and may comprise the use of an assay selected from thegroup consisting of Western blots, enzyme-linked immunosorbent assays,radioimmunoassays, competition immunoassays, dual antibody sandwichassays, immunohistochemical staining assays, agglutination assays, andfluorescent immunoassays. Preferably, said immunologically detecting andquantifying comprises the use of the monoclonal antibody secreted by thehybridoma VU-M75 which has Accession No. ATCC HB 11128. Said detectingand quantifying step could also be envisioned as detecting andquantifying MN nucleic acids, such as, mRNA that expresses MN/CA IX, forexample, using quantitative PCR or other methods known in the art.

REFERENCES

The following references are cited herein or provide updated informationconcerning the MN/CA9 gene and the MN/CA IX protein, and/or cancersassociated with abnormal MN/CA9 gene expression. All the listedreferences as well as other references cited herein are specificallyincorporated by reference.

-   1. Hock et al., J Urol, 167: 57-60 (2002).-   2. Jemal et al., CA-Cancer J Clin, 52: 23-47 (2003).-   3. Takahashi et al., Oncoqene, 22: 6810-6818 (2003).-   4. Higgins et al., Am J Pathol, 162: 925-932 (2003).-   5. Reuter Jr, V. E., and Presti J. C., Semin Oncol: 27: 124-137    (2000).-   6. Linehan, W. M., and Zbar, B., Cancer Cell, 6: 223-228 (2004).-   7. McKiernan et al., Cancer Res, 57: 2362-2365 (1997).-   8. Whittington et al., Proc Natl Acad Sci (USA), 98: 9545-9550    (2001).-   9. Parkkila et al., J Histochem Cytochem, 48: 1601-1608 (2000).-   10. Saarino et al., J Hepatol, 35: 643-649 (2001).-   11. Turner et al., Human Pathol, 28: 740-744 (1997).-   12. Kivela et al., Dig Dis Sci, 46: 2179-2186 (2001).-   13. Pantuck et al., Clin Cancer Res, 9: 4641-4652 (2003).-   14. Potter and Harris, Cell Cycle, 3: 164-167 (2004).-   15. Tripp et al., J Biol Chem, 276: 48615-48618 (2001).-   16. Pastorek et al., Oncoqene, 9: 2877-2888 (1994).-   17. Parkkila S., “An overview of the distribution and function of    carbonic anhydrases in mammals,” In Chegwidden W R, Carter N,    Edwards Y, eds. The carbonic anhydrases: new horizons, Basel,    Switzerland, Birkhauser Verlag, 2000. pp. 76-93.-   18. Murakami et al., BJU Int, 83: 743-747 (1999).-   19. Ivanov et al., Am J Pathol, 158: 905-919 (2001).-   20. Zhuang et al. Mod Pathol, 9: 838-842 (1999).-   21. Ashida et al., J Cancer Res Clin Oncol, 128: 561-568 (2002).-   22. Liao et al., Cancer Res, 57: 2827-2831 (1997).-   23. Ivanov et al., Proc Natl Acad Sci (USA), 95: 12596-12601 (1998).-   24. Maxwell et al., Nature, 399: 271-275 (1999).-   25. Ivan et al., Science, 292: 464-468 (2001).-   26. Jaakkola et al., Science, 292: 468-472 (2001).-   27. Rohzim et al., Cancer Res, 54: 6517-6525 (1994).-   28. Shi et al., Oncogene, 20: 3751-3756 (2001).-   29. Lardner A., J Leukoc Biol, 69: 522-530 (2001).-   30. Teicher et al., Anti Cancer Res, 13: 1549-1556 (1993).-   31. Svastova et al., Exp Cell Res, 290: 332-345 (2003).-   32. Beavon I R G., J Clin Pathol Mol Pathol, 52: 179-187 (1999).-   33. Genda et al., Lab Invest, 80: 387-394 (2000).-   34. Beltran P. J., and Bixby J. L., Front Biosci, 8: 287-299 (2003).-   35. Barnea et al., Mol Cell Biol, 13: 1497-1506 (1993).-   36. Peles et al., Cell, 82: 251-260 (1995).-   37. Potter C P S, and Harris A. L., Brit J Cancer, 89: 2-7 (2003).-   38. Goebel et al., Human Immunol, 63: 813-820 (2002).-   39. Zatovicova et al., J Immunol Meth, 282: 117-134 (2003).-   40. Sun et al., Biochemistry, 41: 6338-6345 (2002).-   41. Dancey, J. E., J Clin Oncol, 22: 2975-2977 (2004).-   42. Vivanco, I., and Sawyers C. L., Nat Rev Cancer, 2: 489-501    (2002).-   43. Vanhaesebroeck et al., Trends Biochem Sci, 22: 267-272 (1997).-   44. Svastova et al., FEBS Lett, 577: 439-445 (2004).-   45. Beasley et al., Cancer Res, 61: 5262-5267 (2001).-   46. Olive et al., Cancer Res, 61: 8924-8929 (2001).-   47. Richard et al., J Biol Chem, 275: 26765-26771 (2000).-   48. Kaluz et al., Cancer Res, 62: 4469-4477 (2002).-   49. Leek et al., Brit J Cancer, 79: 991-995 (1999).-   50. Haddad J. J., and Land S. C., FEBS Lett, 505: 269-274 (2001).-   51. Wykoff et al., Cancer Res, 60: 7075-7083 (2000).-   52. Loncaster et al., Cancer Res, 61: 6394-6399 (2001).-   53. Chia et al., J Clin Oncol, 19: 3660-3668 (2001).-   54. Giatromalonaki et al., Cancer Res, 61: 7992-7998 (2001).-   55. Moch et al., Human Pathol, 28: 1255-1259 (1997).-   56. Uhlman et al., Clin Cancer Res, 1: 913-920 (1995).-   57. Ramp et al., J Urol, 157: 2345-2350 (1997).-   58. Nanjundan et al., J Biol Chem, 278: 37413-37418 (2003).-   59. Chen X., and Resh M. D., J Biol Chem, 277: 49631-49637 (2002).-   60. Schlessinger J., Cell, 103: 211-225 (2000).-   61. Yarden, Y., and Sliwkowski, M. Y., Nat Rev Mol Cell Biol, 2:    127-137 (2001).-   62. Rordorf-Nikolic et al., J Biol Chem, 270: 3662-3666 (1995).-   63. Hellyer et al., Biochem J, 333: 757-763 (1998).-   64. Ponzetto et al., Mol Cell Biol, 13: 4600-4608 (1993).-   65. Wu et al., J Biol Chem, 278: 40425-40428 (2003).-   66. Sekulic A., Cancer Res, 60: 3504-3513 (2000).-   67. Kozma S. C., and Thomas G., BioEssays, 24: 65-71 (2002).-   68. Vogt P. K., Trends Mol Med, 7: 482-484 (2001).-   69. Aoki et al., Proc Natl Acad Sci (USA), 98: 136-141 (2001).-   70. Bjornsti M. A., and Houghton P. J., Nat Rev Cancer, 4: 335-348    (2004).-   71. Abraham R. T., Cell, 111: 9-12 (2002).-   72. Page et al., J Biol Chem, 277: 48403-48409 (2002).-   73. Hudson et al., Mol Cell Biol, 22: 7004-7014 (2002).-   74. Philips et al., J Biol Chem, 280: 22473-22481 (2005).-   75. Sterling et al., J Biol Chem, 277: 25239-25246 (2002).-   76. Mekhail et al., Nat Cell Biol, 6: 642-647 (2004).-   77. Perera et al., Clin Cancer Res, 6: 1518-1523 (2000).-   78. Knebelmann et al., Cancer Res, 58: 226-231 (1998).-   79. Pal et al., J Biol Chem, 272: 27509-27512 (1997).-   80. Kawakami et al., J Biol Chem, 279: 47720-47725 (2004).

ABBREVIATIONS

The following abbreviations are used herein:

-   aa—amino acid-   Akt—protein kinase B (PKB)-   αCA—antibody to CA IX-   ATCC—American Type Culture Collection-   bp—base pairs-   CA—carbonic anhydrase-   ° C.—degrees centigrade-   CA IX-pY—tyrosine phosphorylated CA IX-   DMSO—dimethyl sulfoxide-   ECL—enhanced chemiluminescence method-   EDTA—ethylenediaminetetraacetate-   EGF—epidermal growth factor-   EGFR—epidermal growth factor receptor-   EPO—erythropoietin-   ERK—extracellular signal-regulated kinase-   FBS—fetal bovine serum-   FIH-1—factor inhibiting HIF-1-   HIF—hypoxia-inducible factor-   HRE—hypoxia response element-   HRP—horseradish peroxidase-   IC—intracellular-   IP—immunoprecipitation-   kb—kilobase-   kbp—kilobase pairs-   kd or kDa—kilodaltons-   M—molar-   MAb—monoclonal antibody-   MAPK—mitogen-activated protein kinase-   MBS—MES buffered saline-   MEK—mitogen/extracellular-signal-regulated kinase kinase, also known    as map kinase kinase (MKK)-   MEM—Minimum Essential Medium-   MES—morpholinoethane sulfonic acid-   min.—minute(s)-   mg—milligram-   ml—milliliter-   mM—millimolar-   NEAA—non-essential amino acids-   ng—nanogram-   nm—nanometer-   nM—nanomolar-   NRS—normal rabbit serum-   NSCLC—non-small cell lung cancer-   nt—nucleotide-   ORF—open reading frame-   pAkt—phosphorylated Akt-   PCR—polymerase chain reaction-   PG—proteoglycan-   PI3K—phosphotidylinositol-3-kinase-   pl—isoelectric point-   PMSF—phenylmethyl sulfonyl fluoride-   PTB—phosphotyrosine binding-   P-TYR—phosphotyrosine-   PVDF—polyvinylidene fluoride-   RIPA—radioimmunoprecipitation assay-   RT-PCR—reverse transcription polymerase chain reaction-   SDS—sodium dodecyl sulfate-   SDS-PAGE—sodium dodecyl sulfate-polyacrylamide gel electrophoresis-   TKI—tyrosine kinase inhibitor-   TM—transmembrane-   Tris—tris(hydroxymethyl)aminomethane-   μg—microgram-   μl—microliter-   μM—micromolar-   VEGF—vascular endothelial growth factor-   VEGF-R—vascular endothelial growth factor receptor-   VHL—von Hippel-Lindau protein-   WB—Western blot

Cell Lines

-   CGL3—tumorigenic HeLa x normal fibroblast hybrid cells (HeLa    D98/AH.2 derivative; express CA9, but level increased by both high    density and hypoxia).-   HeLa—aneuploid, epithelial-like cell line isolated from a human    cervical adenocarcinoma [Gey et al., Cancer Res., 12: 264 (1952);    Jones et al., Obstet. Gynecol., 38: 945-949 (1971)] obtained from    Professor B. Korych, [Institute of Medical Microbiology and    Immunology, Charles University; Prague, Czech Republic].-   SKRC-01—human renal cell carcinoma (RCC) cell line overexpressing CA    IX, provided by Dr. Neil Bander [Weill Medical College, Cornell    University, NY]; cell line described in Cho et al., Mol. Carcinog.,    27(3): 184-189 (2000).-   SKRC-08—human renal cell carcinoma (RCC) cell line overexpressing CA    IX, provided by Dr. Neil Bander (supra).-   SKRC-17 human renal cell carcinoma (RCC) cell line which does not    overexpress CA IX, provided by Dr. Neil Bander (supra).

Nucleotide and Amino Acid Sequence Symbols

The following symbols are used to represent nucleotides herein:

Base Symbol Meaning A adenine C cytosine G guanine T thymine U uracil Iinosine M A or C R A or G W A or T/U S C or G Y C or T/U K G or T/U V Aor C or G H A or C or T/U D A or G or T/U B C or G or T/U N/X A or C orG or T/U

There are twenty main amino acids, each of which is specified by adifferent arrangement of three adjacent nucleotides (triplet code orcodon), and which are linked together in a specific order to form acharacteristic protein. A three-letter or one-letter convention may beused herein to identify said amino acids as follows:

3 Ltr. 1 Ltr. Amino acid name Abbrev. Abbrev. Alanine Ala A Arginine ArgR Asparagine Asn N Aspartic Acid Asp D Cysteine Cys C Glutamic Acid GluE Glutamine Gln Q Glycine Gly G Histidine His H Isoleucine Ile I LeucineLeu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro PSerine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine ValV Unknown or other X

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A—C depicts EGF dependent phosphorylation of membrane associatedcarbonic anhydrase IX. SKRC-01 cells were serum starved and stimulatedwith increasing concentrations of EGF for 30 min. Whole cell lysateswere immunoprecipitated (IP) with polyclonal antibody to CA IX (CA) andthe blots (WB) were probed with a monoclonal antibody to phosphotyrosine(P-TYR) (panel A). As a negative control, the same experiment wasrepeated with normal rabbit serum (NRS) instead of the polyclonalantibody to CA IX, shown in panel B. Panel C shows that equivalentamounts of protein were loaded when the same amounts of protein loadedfor panel A were run on another gel and probed with the monoclonalantibody to CA IX (M75 MAb). FIG. 1D shows kinetics of the loss ofphosphorylation of CA IX (CA IX-pY) in EGF stimulated SKRC-01 cellsusing conditions as in FIG. 1A-C. The initial EGF stimulus was removedafter 30 min and the loss of CA IX-pY signal was followed up to 90 min.

FIG. 2 depicts co-immunoprecipitation of p85 subunit of PI3K with thetyrosine phosphorylated CA IX. SKRC-01 cells stimulated in the presenceor absence of EGF were solubilized and immunoprecipitated (IP) with M75MAb to CA IX. The blots (WB) were probed with a polyclonal antibody tothe p85 regulatory subunit of PI3K (upper panel lanes 1, 2 and 3). As aloading control, identical blots were probed for the presence of thetotal p85 subunit of PI3K (lower panel). In some cases, the lipid raftmembrane fractions from EGF stimulated SKRC-01 cells were solubilizedand processed for immunoprecipitation with CA IX and immunoblotting withp85 (upper panel, lane 4).

FIG. 3 shows the effect of pharmacological PI3K inhibition on thephosphorylation status of Akt. SKRC-17 cells (CA IX negative) weretransiently transfected with the plasmid pSG5C-wtCAIX and serum starvedbefore the start of the study. The cells were pretreated for 8 h withwortmannin and LY294002 at the indicated concentrations. At the end ofthis pretreatment period, the cells were stimulated with EGF and wholecell extracts were made with radioimmunoprecipitation (RIPA) buffer.Immunoblot assays were performed using antibodies against phosphorylatedAkt (for serine 473 and threonine 308). As a measure of the loadingcontrols, blots were probed for total unphosphorylated Akt protein,shown in panel B. Preliminary work with either vehicle only controls(DMSO) or EGF unstimulated controls showed no phosphorylation of eitherSer 473 or Thr 308 in serum starved conditions (data not shown). Underthe EGF stimulated conditions, the phosphorylation of Akt at Thr 308 wasnot observed (panel C).

FIG. 4A depicts the effect of serum starvation and EGF stimulation instably transfected SKRC-17 cells expressing CA IX as seen by thedifferences in Akt phosphorylation. Upper panel: immunoblot using thephosphospecific Akt (Ser 473) antibody as probe; Lower panel, anidentical blot using antibody for the unphosphorylated Akt protein asprobe, which also serves as a loading control. Lane 1: SKRC-17 cellstransfected with empty vector pSG5C and pcDNA3.1 in the presence of EGF(50 ng/ml); lane 2: cells expressing wt CA IX in the presence of EGF;lane 3: G418 resistant cells expressing the wild-type CA IX in theabsence of EGF (i.e., serum starvation); lane 4: CA IX YF mutantexpressing cells in the presence of EGF and lane 5: CA IX YF mutantexpressing cells in the absence of EGF.

FIG. 4B shows the correlation of Akt phosphorylation with the level ofexpression of CA IX and HIF-1 protein subunits under normoxic conditionsin SKRC cells. SKRC-17 (CA IX negative), -08 (low expression of CA IX)and -01 cells (high expression of CA IX) were serum starved andstimulated with 50 ng/ml EGF as described earlier. Thirty minutes poststimulus, cell lysates were prepared, subjected to denaturing gelelectrophoresis and immunoblots were probed for the expression of CA IX(panel A), total unphosphorylated Akt (panel B), phosphoAkt (Ser 473,panel C), HIF-1α subunit (panel D) and HIF-1β subunit (panel E) usingspecific antibodies. The signals were developed using the correspondingsecondary antibodies conjugated with horse radish peroxidase (HRP) andenhanced chemiluminescence (ECL). Panels B and E also served as loadingcontrols.

FIG. 5 provides a diagrammatic sketch of the major signaling pathways inclear cell carcinoma cell placing CA IX in the lipid rafts where it canget phosphorylated in a growth factor dependent manner and participatein the signaling processes regulated by PI3K and mammalian target ofrapamycin (mTOR). The CA IX protein recruited to the lipid rafts isdepicted in its dimerized form. This figure also depicts the establishedrole of PI3K as a mediator of several survival, proliferation andapoptosis resistance pathways that lead to resistance tochemotherapeutic drugs. The major role of mTOR as an integrator ofseveral signaling inputs is also presented with particular reference tocap-dependent translation of target proteins that include cyclin D1,c-myc and most importantly, HIF-1α. The placement of CA IX tyrosinephosphorylation in the midst of these cell signaling systems forms thebasis of a vicious cycle, whereby CA IX mediated activation of Aktpromotes the expression of HIF-Iα which in turn promotes the expressionof CA IX leading to poor prognosis in advanced cases of clear cell RCC.Increased acidification of the extracellular compartment contributes toincreased invasive potential. The HIF-α target gene VEGF contributes toenhanced angiogenesis which is one of the hallmarks of clear cell RCC.

FIG. 6A-C provides the nucleotide sequence for a MN cDNA [SEQ ID NO: 1]clone isolated. FIG. 6 A-C also sets forth the predicted amino acidsequence [SEQ ID NO: 2] encoded by the cDNA.

FIG. 7 provides an exon-intron map of the human MN/CA IX gene. Thepositions and sizes of the exons (numbered, cross-hatched boxes), Alurepeat elements (open boxes) and an LTR-related sequence (firstunnumbered stippled box) are adjusted to the indicated scale. The exonscorresponding to individual MN/CA IX protein domains are enclosed indashed frames designated PG (proteoglycan-like domain), CA (carbonicanhydrase domain), TM (transmembrane anchor) and IC (intracytoplasmictail).

DETAILED DESCRIPTION

The MN/CA IX protein, via its CA and PG domains, is functionallyimplicated in tumorigenesis as part of the regulatory mechanisms thatcontrol pH and cell adhesion. However, it had been previously unknownwhether MN/CA IX's IC domain had a potential tumorigenic role as well.

As shown in the Examples below, the inventor found that in RCC celllines the sole tyrosine moiety present in CA IX's IC region can bephosphorylated via the EGFR pathway, which interacts with the p85subunit of PI3K to activate Akt, which activation is implicated incancer progression. That finding of that apparent third tumorigenic rolefor MN indicates that there is a positive feedback loop for CA9expression in RCC, mediated by the PI3K pathway, which may contribute tothe aggressiveness of RCC and potentially other preneoplastic/neoplasticdiseases associated with abnormal MN/CA IX expression.

Preneoplastic/Neoplastic Tissues

The novel methods of the present invention concern treatingpreneoplastic/neoplastic diseases by preventing phosphorylation of CAIX's IC domain using EGFR pathway inhibitors, alone or in combinationwith MN-directed therapies. Those methods are expected to be effectivefor any preneoplastic/neoplastic disease characterized by abnormalMN/CA9 gene expression. Exemplary preneoplastic/neoplastic diseasesinclude, among other preneoplastic/neoplastic diseases known to beassociated with abnormal MN expression, at the leastpreneoplastic/neoplastic diseases selected from the group consisting ofmammary, urinary tract, bladder, kidney, ovarian, uterine, cervical,endometrial, squamous cell, adenosquamous cell, vaginal, vulval,prostate, liver, lung, skin, thyroid, pancreatic, testicular, brain,head and neck, mesodermal, sarcomal, stomach, spleen, gastrointestinal,esophageal, colorectal and colon preneoplastic/neoplastic diseases.

As used herein, “cancerous” and “neoplastic” have equivalent meanings,and “precancerous” and “preneoplastic” have equivalent meanings.

MN Gene and Protein

The terms “CA IX” and “MN/CA9” are herein considered to be synonyms forMN. Also, the G250 antigen is considered to refer to MNprotein/polypeptide [Jiang et al., PNAS (USA) 97: 1749-173 (2000)].

Zavada et al., WO 93/18152 and/or WO 95/34650 disclose the MN cDNAsequence shown herein in FIGS. 6A-6C [SEQ ID NO: 1], the MN amino acidsequence [SEQ ID NO: 2] also shown in FIGS. 6A-6C, and the MN genomicsequence [SEQ ID NO: 3]. The MN gene is organized into 11 exons and 10introns.

The ORF of the MN cDNA shown in FIG. 6 has the coding capacity for a 459amino acid protein with a calculated molecular weight of 49.7 kd. Theoverall amino acid composition of the MN protein is rather acidic, andpredicted to have a pl of 4.3. Analysis of native MN protein from CGL3cells by two-dimensional electrophoresis followed by immunoblotting hasshown that in agreement with computer prediction, the MN is an acidicprotein existing in several isoelectric forms with pls ranging from 4.7to 6.3.

The first thirty seven amino acids of the MN protein shown in FIGS.6A-6C is the putative MN signal peptide [SEQ ID NO: 4]. The MN proteinhas an extracellular domain [amino acids (aa) 38-414 of FIGS. 6A-6C; SEQID NO: 5], a transmembrane domain [aa 415-434; SEQ ID NO: 6] and anintracellular domain [aa 435-459; SEQ ID NO: 7]. The extracellulardomain contains the proteoglycan-like domain [aa 53-111: SEQ ID NO: 8]and the carbonic anhydrase (CA) domain [aa 135-391; SEQ ID NO: 9].

The CA domain is essential for induction of anchorage independence,whereas the TM anchor and IC tail are dispensable for that biologicaleffect. The MN protein is also capable of causing plasma membraneruffling in the transfected cells and appears to participate in theirattachment to the solid support. The data evince the involvement of MNin the regulation of cell proliferation, adhesion and intercellularcommunication.

MN Gene—Cloning and Sequencing

FIG. 6A-C provides the nucleotide sequence for a full-length MN cDNAclone [SEQ ID NO: 1]. A complete MN genomic sequence is represented bySEQ ID NO: 3, and the nucleotide sequence for a proposed MN promoter isrepresented by SEQ ID NO: 24.

It is understood that because of the degeneracy of the genetic code,that is, that more than one codon will code for one amino acid [forexample, the codons TTA, TTG, CTT, CTC, CTA and CTG each code for theamino acid leucine (leu)], that variations of the nucleotide sequencesin, for example, SEQ ID NOS: 1 and 3 wherein one codon is substitutedfor another, would produce a substantially equivalent protein orpolypeptide according to this invention. All such variations in thenucleotide sequences of the MN cDNA and complementary nucleic acidsequences are included within the scope of this invention.

It is further understood that the nucleotide sequences herein describedand shown in FIG. 6 represent only the precise structures of the cDNA,genomic and promoter nucleotide sequences isolated and described herein.It is expected that slightly modified nucleotide sequences will be foundor can be modified by techniques known in the art to code forsubstantially similar or homologous MN proteins and polypeptides, forexample, those having similar epitopes, and such nucleotide sequencesand proteins/polypeptides are considered to be equivalents for thepurpose of this invention.

DNA or RNA having equivalent codons is considered within the scope ofthe invention, as are synthetic nucleic acid sequences that encodeproteins/polypeptides homologous or substantially homologous to MNproteins/polypeptides, as well as those nucleic acid sequences thatwould hybridize to said exemplary sequences [SEQ. ID. NOS. 1, 3 and 24]under stringent conditions, or that, but for the degeneracy of thegenetic code would hybridize to said cDNA nucleotide sequences understringent hybridization conditions. Modifications and variations ofnucleic acid sequences as indicated herein are considered to result insequences that are substantially the same as the exemplary MN sequencesand fragments thereof.

Only very closely related nt sequences having a homology of at least80-90%, preferably at least 90%, would hybridize to each other understringent conditions. A sequence comparison of the MN cDNA sequenceshown in FIG. 6 and a corresponding cDNA of the human carbonic anhydraseII (CA II) showed that there are no stretches of identity between thetwo sequences that would be long enough to allow for a segment of the CAII cDNA sequence having 25 or more nucleotides to hybridize understringent hybridization conditions to the MN cDNA or vice versa.

Stringent hybridization conditions are considered herein to conform tostandard hybridization conditions understood in the art to be stringent.For example, it is generally understood that stringent conditionsencompass relatively low salt and/or high temperature conditions, suchas provided by 0.02 M to 0.15 M NaCl at temperatures of 50° C. to 70° C.Less stringent conditions, such as, 0.15 M to 0.9 M salt at temperaturesranging from 20° C. to 55° C. can be made more stringent by addingincreasing amounts of formamide, which serves to destabilize hybridduplexes as does increased temperature.

Exemplary stringent hybridization conditions are described in Sambrooket al., Molecular Cloning: A Laboratory Manual, pages 1.91 and 9.47-9.51(Second Edition, Cold Spring Harbor Laboratory Press; Cold SpringHarbor, N.Y.; 1989); Maniatis et al., Molecular Cloning: A LaboratoryManual, pages 387-389 (Cold Spring Harbor Laboratory; Cold SpringHarbor, N.Y.; 1982); Tsuchiya et al., Oral Surgery, Oral Medicine, OralPathology, 71(6): 721-725 (June 1991); and in U.S. Pat. No. 5,989,838,U.S. Pat. No. 5,972,353, U.S. Pat. No. 5,981,711, and U.S. Pat. No.6,051,226.

Plasmids containing the MN genomic sequence (SEQ ID NO: 3)—the A4a cloneand the XE1 and XE3 subclones—were deposited at the American TypeCulture Collection (ATCC) on Jun. 6, 1995, respectively under ATCCDeposit Nos. 97199, 97200, and 97198.

MN Proteins and Polypeptides

The phrase “MN proteins and/or polypeptides” (MN proteins/polypeptides)is herein defined to mean proteins and/or polypeptides encoded by an MNgene or fragments thereof. An exemplary and preferred MN proteinaccording to this invention has the deduced amino acid sequence shown inFIG. 6. Preferred MN proteins/polypeptides are those proteins and/orpolypeptides that have substantial homology with the MN protein shown inFIG. 6. For example, such substantially homologous MNproteins/polypeptides are those that are reactive with the MN-specificantibodies, preferably the Mab M75 or its equivalent. The VU-M75hybridoma that secretes the M75 Mab was deposited at the ATCC under HB11128 on Sep. 17, 1992.

A “polypeptide” or “peptide” is a chain of amino acids covalently boundby peptide linkages and is herein considered to be composed of 50 orless amino acids. A “protein” is herein defined to be a polypeptidecomposed of more than 50 amino acids. The term polypeptide encompassesthe terms peptide and oligopeptide.

It can be appreciated that a protein or polypeptide produced by aneoplastic cell in vivo could be altered in sequence from that producedby a tumor cell in cell culture or by a transformed cell. Thus, MNproteins and/or polypeptides which have varying amino acid sequencesincluding without limitation, amino acid substitutions, extensions,deletions, truncations and combinations thereof, fall within the scopeof this invention. It can also be appreciated that a protein extantwithin body fluids is subject to degradative processes, such as,proteolytic processes; thus, MN proteins that are significantlytruncated and MN polypeptides may be found in body fluids, such as,sera. The phrase “MN antigen” is used herein to encompass MN proteinsand/or polypeptides.

It will further be appreciated that the amino acid sequence of MNproteins and polypeptides can be modified by genetic techniques. One ormore amino acids can be deleted or substituted. Such amino acid changesmay not cause any measurable change in the biological activity of theprotein or polypeptide and result in proteins or polypeptides which arewithin the scope of this invention, as well as, MN muteins.

Preparation of MN-Specific Antibodies

The term “antibodies” is defined herein to include not only wholeantibodies but also biologically active fragments of antibodies,preferably fragments containing the antigen binding regions. Furtherincluded in the definition of antibodies are bispecific antibodies thatare specific for MN protein and to another tissue-specific antigen,preferably bispecific antibodies that are specific for MN protein andEGFR.

Antibodies useful according to the methods of the invention may beprepared by conventional methodology and/or by genetic engineering.Antibody fragments may be genetically engineered, preferably from thevariable regions of the light and/or heavy chains (V_(H) and V_(L)),including the hypervariable regions, and still more preferably from boththe V_(H) and V_(L) regions. For example, the term “antibodies” as usedherein includes polyclonal and monoclonal antibodies and biologicallyactive fragments thereof including among other possibilities “univalent”antibodies [Glennie et al., Nature, 295: 715 (1982)]; Fab proteinsincluding Fab′ and F(ab)₂ fragments whether covalently or non-covalentlyaggregated; light or heavy chains alone, preferably variable heavy andlight chain regions (V_(H) and V_(L) regions), and more preferablyincluding the hypervariable regions [otherwise known as thecomplementarity determining regions (CDRs) of the V_(H) and V_(L)regions]; F_(C) proteins; “hybrid” antibodies capable of binding morethan one antigen; constant-variable region chimeras; “composite”immunoglobulins with heavy and light chains of different origins;“altered” antibodies with improved specificity and other characteristicsas prepared by standard recombinant techniques and alsooligonucleotide-directed mutagenesis techniques [Dalbadie-McFarland etal., PNAS (USA, 79: 6409 (1982)].

For many uses, particularly for pharmaceutical uses or for in vivotracing, partially or more preferably fully humanized antibodies and/orbiologically active antibody fragments may be found most particularlyappropriate. Such humanized antibodies/antibody fragments can beprepared by methods well known in the art.

The antibodies useful according to this invention to identify MNproteins/polypeptides can be labeled in any conventional manner, forexample, with enzymes such as horseradish peroxidase (HRP), fluorescentcompounds, or with radioactive isotopes such as, ¹²⁵I, among otherlabels. A preferred label, according to this invention is ¹²⁵I, and apreferred method of labeling the antibodies is by using chloramine-T[Hunter, W. M., “Radioimmunoassay,” In: Handbook of ExperimentalImmunology, pp. 14.1-14.40 (D. W. Weir ed.; Blackwell,Oxford/London/Edinburgh/Melbourne; 1978)]. Other exemplary labels mayinclude, for example, allophycocyanin and phycoerythrin, among manyother possibilities.

Representative monoclonal antibodies useful according to this inventioninclude Mabs M75, MN9, MN12 and MN7 described in earlier Zavada et al.patents and patent applications. [U.S. Pat. No. 6,297,041; U.S. Pat. No.6,204,370; U.S. Pat. No. 6,093,548; U.S. Pat. No. 6,051,226; U.S. Pat.No. 6,004,535; U.S. Pat. No. 5,989,838; U.S. Pat. No. 5,981,711; U.S.Pat. No. 5,972,353; U.S. Pat. No. 5,955,075; U.S. Pat. No. 5,387,676; USApplication Nos: 20050031623, 20030049828, and 20020137910; andInternational Publication No. WO 03/100029]. Monoclonal antibodiesuseful according to this invention serve to identify MNproteins/polypeptides in various laboratory prognostic tests, forexample, in clinical samples. For example, monoclonal antibody M75 (MabM75) is produced by mouse lymphocytic hybridoma VU-M75, which wasdeposited under ATCC designation HB 11128 on Sep. 17, 1992 at theAmerican Tissue Type Culture Collection [ATCC]. The production ofhybridoma VU-M75 is described in Zavada et al., InternationalPublication No. WO 93/18152. Mab M75 recognizes both the nonglycosylatedGST-MN fusion protein and native MN protein as expressed in CGL3 cellsequally well. The M75 Mab recognizes both native and denatured forms ofthe MN protein [Pastorekova et al., Virology, 187: 620-626 (1992)].

General texts describing additional molecular biological techniquesuseful herein, including the preparation of antibodies include Bergerand Kimmel, Guide to Molecular Cloning Techniques, Methods inEnzymology, Vol. 152, Academic Press, Inc. (1987); Sambrook et al.,Molecular Cloning: A Laboratory Manual, (Second Edition, Cold SpringHarbor Laboratory Press; Cold Spring Harbor, N.Y.; 1989) Vols. 1-3;Current Protocols in Molecular Biology, F. M. Ausabel et al. [Eds.],Current Protocols, a joint venture between Green Publishing Associates,Inc. and John Wiley & Sons, Inc. (supplemented through 2000); Harlow etal., Monoclonal Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory Press (1988), Paul [Ed.]; Fundamental Immunology, LippincottWilliams & Wilkins (1998); and Harlow et al., Using Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory Press (1998).

Epitopes

The affinity of a MAb to peptides containing an epitope depends on thecontext, e.g. on whether the peptide is a short sequence (4-6 aa), orwhether such a short peptide is flanked by longer aa sequences on one orboth sides, or whether in testing for an epitope, the peptides are insolution or immobilized on a surface. Therefore, it would be expected byones of skill in the art that the representative epitopes describedherein for the MN-specific MAbs would vary in the context of the use ofthose MAbs.

The term “corresponding to an epitope of an MN protein/polypeptide” willbe understood to include the practical possibility that, in someinstances, amino acid sequence variations of a naturally occurringprotein or polypeptide may be antigenic and confer protective immunityagainst neoplastic disease and/or anti-tumorigenic effects. Possiblesequence variations include, without limitation, amino acidsubstitutions, extensions, deletions, truncations, interpolations andcombinations thereof. Such variations fall within the contemplated scopeof the invention provided the protein or polypeptide containing them isimmunogenic and antibodies elicited by such a polypeptide or proteincross-react with naturally occurring MN proteins and polypeptides to asufficient extent to provide protective immunity and/or anti-tumorigenicactivity when administered as a vaccine.

Immunodominant Epitopes in PG Domain and In Neighboring Regions

As indicated above, the extracellular domain of the full-length CA IXcomprises the PG and CA domains as well as some spacer or perhaps hingeregions. The CA IX immunodominant epitopes are primarily in the PGregion at about aa 53-111 (SEQ ID NO: 8) or at about aa 52-125 [SEQ IDNO: 31], preferably now considered to be at about aa 52-125 [SEQ ID NO:31]. The immunodominant epitopes of CA IX may be located in regionsneighboring the PG region. For example, the epitope for aa 36-51 (SEQ IDNO: 21) would be considered an immunodominant epitope.

The main CA IX immunodominant epitope is that for the M75 MAb. The M75monoclonal antibody is considered to be directed to an immunodominantepitope in the N-terminal, proteoglycan-like (PG) region of CA IX.Alignment of amino acid sequences illustrates significant homologybetween the MN/CA IX protein PG region (aa 53-111) [SEQ ID NO: 8] andthe human aggrecan (aa 781-839) [SEQ ID NO: 10]. The epitope of M75 hasbeen identified as amino acid sequence PGEEDLP (SEQ ID NO: 11), which is4× identically repeated in the N-terminal PG region of CA IX [Zavada etal. (2000)]. Closely related epitopes to which the M75 MAb may alsobind, which are also exemplary of immunodominant epitopes include, forexample, the immunodominant 6× tandem repeat that can be found at aminoacids (aa) 61-96 (SEQ ID NO. 12) of FIG. 6A-6C, showing the predicted CAIX amino acid sequence. Variations of the immunodominant tandem repeatepitopes within the PG domain include GEEDLP (SEQ ID NO: 13) (aa 61-66,aa 79-84, aa 85-90 and aa 91-96), EEDL (SEQ ID NO: 14) (aa 62-65, aa80-83, aa 86-89, aa 92-95), EEDLP (SEQ ID NO: 15) (aa 62-66, aa 80-84,aa 86-90, aa 92-96), EDLPSE (SEQ ID NO: 16) (aa 63-68), EEDLPSE (SEQ IDNO: 17) (aa 62-68), DLPGEE (SEQ ID NO: 18) (aa 82-87, aa 88-93), EEDLPS(SEQ ID NO: 19) (aa 62-67) and GEDDPL (SEQ ID NO: 20) (aa 55-60). Otherimmunodominant epitopes could include, for example, aa 68-91 (SEQ ID NO:22).

The monoclonal antibodies MN9 and MN12 are considered to be directed toimmunodominant epitopes within the N-terminal PG region SEQ ID NOS:19-20, respectively. The MN7 monoclonal antibody could be directed to animmunodominant epitope neighboring the PG region at aa 127-147 (SEQ IDNO: 23) of FIG. 6A-6C.

An epitope considered to be preferred within the CA domain (SEQ ID NO:9) is from about aa 279-291. An epitope considered to be preferredwithin the intracellular domain (IC domain) (SEQ ID NO: 7) is from aboutaa 435-450. An exemplary preferred MN-specific antibody thatspecifically binds the carbonic anhydrase domain of MN protein is theV/10 Mab, which is produced by the hybridoma VU-V/10, deposited atBCCM™/LMBP in Ghent, Belgium under Accession No. LMBP 6009CB.

Assays Assays to Screen for MN/CA9 Gene Expression in Tissues

The methods may comprise screening for MN/CA9 gene expression product,if any, present in a sample taken from a patient diagnosed withpreneoplastic/neoplastic disease; the MN/CA9 gene expression product canbe MN protein, MN polypeptide, mRNA encoding a MN protein orpolypeptide, a cDNA corresponding to an mRNA encoding a MN protein orpolypeptide, or the like. If the MN/CA9 gene expression product ispresent at abnormal levels in said sample, the patient may be a suitablecandidate for the therapeutic methods of the invention. In most cases,the abnormal levels would be increased MN/CA9 expression levels intissues that do not normally express MN.

In a preferred embodiment of the invention, the MN gene expressionproduct is MN antigen, and the presence or absence of MN antigen isscreened in preneoplastic/neoplastic mammalian samples, preferably humansamples. Such preneoplastic/neoplastic samples can be tissue specimens,tissue extracts, cells, cell lysates and cell extracts, among othersamples. Preferred tissue samples are formalin-fixed, paraffin-embeddedtissue samples or frozen tissue samples.

It can be appreciated by those of skill in the art that various otherpreneoplastic/neoplastic samples can be used to screen for the MN geneexpression products. For example, in the case of a patient afflictedwith a neoplastic disease, wherein the disease is a tumor, the samplemay be taken from the tumor or from a metastatic lesion derived from thetumor.

It can further be appreciated that alternate methods, in addition tothose disclosed herein, can be used to quantify the MN gene expressionproducts.

In preferred embodiments, the gene expression product is MN antigenwhich is detected by immunohistochemical staining (e.g., using tissuearrays or the like). Preferred tissue specimens to assay byimmunohistochemical staining, for example, include cell smears,histological sections from biopsied tissues or organs, and imprintpreparations among other tissue samples. Such tissue specimens can bevariously maintained, for example, they can be fresh, frozen, orformalin-, alcohol- or acetone- or otherwise fixed and/orparaffin-embedded and deparaffinized. Biopsied tissue samples can be,for example, those samples removed by aspiration, bite, brush, cone,chorionic villus, endoscopic, excisional, incisional, needle,percutaneous punch, and surface biopsies, among other biopsy techniques.

Many formats can be adapted for use with the methods of the presentinvention. The detection and quantitation of MN protein or MNpolypeptide can be performed, for example, by Western blots,enzyme-linked immunosorbent assays, radioimmunoassays, competitionimmunoassays, dual antibody sandwich assays, immunohistochemicalstaining assays, agglutination assays, fluorescent immunoassays,immunoelectron and scanning microscopy using immunogold, among otherassays commonly known in the art. The detection of MN gene expressionproducts in such assays can be adapted by conventional methods known inthe art.

It is also apparent to one skilled in the art of immunoassays that MNproteins or polypeptides can be used to detect and quantitate MN antigenin body tissues and/or cells of patients. In one such embodiment, animmunometric assay may be used in which a labelled antibody made to MNprotein is used. In such an assay, the amount of labelled antibody whichcomplexes with the antigen-bound antibody is directly proportional tothe amount of MN antigen in the sample.

Methods of EGFR-Directed Cancer Therapy Based on Detection of AbnormalMN Expression EGFR Pathway Inhibitors

As indicated above, the invention is based upon the discovery that thesole tyrosine moiety in the IC region of CA IX can be phosphorylated inan EGFR-dependent manner and result in activation of Akt, whichactivation is implicated in cancer progression. Therefore, EGFR pathwayinhibitors can be used therapeutically to treat preneoplastic/neoplasticdiseases characterized by abnormal MN/CA9 gene expression.

As used herein, “EGFR pathway inhibitors” include any therapies that aretargeted to the EGFR pathway, including targeting any of the EGFRcomponents. Preferred therapies that target the EGFR pathway includeEGFR-specific antibodies, EGFR tyrosine kinase inhibitors, and otherEGFR-targeted agents [recently reviewed in Marshall, Cancer, 107(6):1207-1218 (2006)].

Monoclonal antibodies (MAbs) block the extracellular ligand-bindingportion of the EGFR and interfere with its activation. Exemplary EGFRpathway inhibitors that have been approved by the U.S. Food and DrugAdministration include cetuximab (IMC-225, Erbitux™; ImClone Systems,Princeton, N.J.), a monoclonal antibody for the treatment of colorectalcancer. Other exemplary EGFR-specific Mabs that are undergoing clinicaltesting are panitumumab (ABX-EGF; Abgenix, Fremont, Calif.), nimotuzumab(TheraCIM™, h-R3; CIMYM Biosciences, Ontario, Canada), matuzumab(EMD-72000; EMD Pharmaceuticals/Merck KgaA); MDX-447, a dual EGFR andCD64 inhibitor (HuMax™-EGFr; Medarex, Princeton, N.J.), and Mab-806(Ludwig Institute, Victoria, Australia).

In contrast, tyrosine kinase inhibitors (TKIs) block induction of theintracellular tyrosine kinase-mediated signaling pathways by binding ator near the ATP binding site on the intracellular kinase domain.Exemplary small-molecule EGFR TKIs that have received U.S. FDA approvalare erlotinib (Tarceva®, OSI-774; CP-358,774; OSI Pharmaceuticals incollaboration with Genentech and Roche pharmaceuticals) for thetreatment of NSCLC and pancreatic cancer, and gefitinib (Iressa®,ZD1839; AstraZeneca, Wilmington, Del.) for NSCLC. Cenertinib (CI-1033;Pfizer Pharmaceuticals, Groton, Conn.) is an irreversible, pan-ErbBinhibitor of receptor tyrosine kinase phosphorylation that has undergonePhase I studies. Additional exemplary TKIs that are in Phase I and/orPhase II clinical trials are the irreversible EGFR and HER-2 dualinhibitor EKB-569 (Wyeth-Ayerst, Madison, N.J.); and threedual-EGFR/ErbB-2-reversible EGFR TKIs, PKI-166 (Novartis International,Basel, Switzerland), GW572016 (GlaxoSmithKline, Research Triangle Park,N.C.), and ARRY-334543 (Array BioPharma, CO). Still other TKIs currentlyin preclinical development are PD153035 and PD158780 (Parke-Davis, AnnArbor, Mich.).

Use of EGFR Inhibitors with Conventional or MN-Directed Therapies

According to the methods of the invention, the EGFR inhibitors can becombined with MN/CA IX-specific antibodies and a variety of conventionaltherapeutic drugs, different inhibitors of cancer-related pathways,bioreductive drugs, and/or radiotherapy, wherein different combinationsof treatment regimens with the EGFR inhibitors may increase overalltreatment efficacy. Preferred therapies to be used in combination withEGFR inhibitors are inhibitors of the PI3K pathway and/or the MAPKpathway, as well as MN-directed therapies.

PI3K Pathway Inhibitors

Activation of the phosphotidylinositol-3-kinase (PI3K)/Akt cell survivalpathway in many cancers, and its newly-discovered association with CAIX's IC domain, makes it an obvious target for cancer therapy. Becausethis pathway also has an important role in the survival of normal cells,however, it is important to achieve cancer selectivity; thecancer-selective proapoptotic protein Par-4 is a key target forinactivation by PI3K/Akt signaling [Goswami et al., Cancer Res., 66(6):2889-2892 (2006)]. Several anticancer therapies target, albeitindirectly, the PI3K/Akt pathway and cause inhibition of Akt1phosphorylation and induction of apoptosis. Examples include Herceptin®,which inhibits ErbB-2 in breast cancer cells; cyclooxygenase-2 (COX-2)inhibitors, which inhibit COX-2 and PD1 in colon and prostate cancer;and imatinib mesylate (Gleevec, STI-571), which targets bcr-abl inleukemia.

MAPK Inhibitors

As used herein, “MAPK pathway inhibitors” include any therapies that aretargeted to the MAPK pathway, including targeting any of the MAPKcomponents, such as, Ras, Raf, MEK, and ERK, including but not limitedto inhibition of their protein expression (e.g., antisenseoligonucleotides), prevention of membrane localization essential forMAPK activation, and inhibition of downstream effectors of MAPK (e.g.,Raf serine/threonine kinases) [for review of MAPK inhibitors, see Gollobet al., Semin Oncol., 33(4): 392-406 (2006)]. MAPK pathway-directedtherapies include but are not limited to multi-kinase inhibitors,tyrosine kinase inhibitors, monoclonal antibodies, as well asbiologically active antibody fragments, polyclonal antibodies, andanti-anti-idiotype antibodies and related antibody based therapies,bis-aryl ureas, and omega-carboxypyridyl substituted ureas and the like.Preferred MAPK pathway inhibitors are Raf kinase inhibitors, which aredescribed in more detail below

Thus far, the most successful clinical drugs targeting theRas/Raf/MEK/ERK cascade appear to be those that target Raf [Schreck andRapp, Int. J. Cancer, 119: 2261-2271 (2006)], including the multi-kinaseinhibitor Sorafenib (BAY 43-9006), and antisense and heat shock protein90 (HSP90) inhibitors.

An exemplary and preferred MAPK pathway-directed therapy according tothe invention is the bis-aryl urea Sorafenib (BAY 43-9006) [Nexavar®;Onyx Pharmaceuticals, Richmond, Calif. (USA), and Bayer Corporation,West Haven, Conn. (USA); Wilhelm and Chien, Curr Pharm Des, 8: 2255-2257(2002); Wilhelm et al., Cancer Res., 64: 7099-7109 (2004); Strumberg, D,Drugs Today (Barc), 41: 773-84, 2005; Lyons et al., Endocrine-RelatedCancer, 8: 219-225 (2001)], a small molecule and novel dual-actioninhibitor of both Raf (a protein-serine/threonine kinase) and VEGFR(vascular endothelial growth factor receptor, a receptor tyrosinekinase), and consequently an inhibitor of both tumor cell proliferationand angiogenesis. In addition, Sorafenib has been found to inhibitseveral other receptor tyrosine kinases involved in tumor progressionand neovascularization, including PDGFR-β, Flt-3, and c-KIT. In December2005 Sorafenib was approved by the FDA for patients with advanced renalcell carcinoma (RCC). Other preferred MAPK-directed therapies accordingto the invention are omega-carboxypyridyl substituted ureas, which arederivatives of bis-aryl ureas with improved solubility in water [Khireet al., Bioorg Med Chem. Lett., 14(3): 783-786 (2004)].

Other exemplary therapies that target the MAPK pathway include MEKinhibitors. PD-0325901 (Pfizer) and ARRY-142886 (AZD-6244, Array andAstraZeneca) are small-molecule inhibitors currently in clinicaldevelopment [Gollob et al., Semin Oncol., 33(4): 392-406 (2006); Doyleet al., Proc Am Soc Clin Oncol, 24: 3075, 2005 (Abstr.); Lee et al.,Cancer, 2: 368 (2004) (Suppl.)] Those two orally available agents arenon-ATP competitive allosteric inhibitors of MEK, which unlike themajority of ATP-competitive analogs, show high selectivity for MEK inbiochemical assays. PD-0325901 is a second-generation compound derivedfrom CI-1040 (PD184352, Pfizer), an oral MEK inhibitor which began PhaseII clinical trials. PD-0325901 has an IC₅₀ value 200-fold lower thanCI-1040, and is also more soluble with improved metabolic stability andbioavailability. PD-0325901 and ARRY-142886 have shown potent anti-tumoractivity in tumor xenograft models. ARRY-142886 is currently beingevaluated in phase 1 trials, while phase I/II clinical trial findingshave recently been reported with PD-0325901.

MN-Directed Therapies

Because of MN protein's unique characteristics, it is an attractivecandidate target for cancer therapy. In comparison to othertumor-related molecules (e.g. growth factors and their receptors), MNhas the unique property of being differentially expressed inpreneoplastic/neoplastic and normal tissues. Because of the extremelylimited expression of MN protein in normal tissues, chemotherapeuticagents that target its expression would be expected to have reduced sideeffects, relative to agents that target proteins more extensively foundin normal tissues (e.g., tamoxifen which binds the estrogen receptor,and finasteride which binds the androgen receptor). Furthermore, Phase Iand II clinical trials of an MN-specific drug, Rencarex®, have shownthat at least one MN-specific agent is well-tolerated, with no seriousdrug-related side effects, further supporting MN as a possible targetfor cancer chemotherapy.

Many MN-directed therapies may be useful according to the methods of thepresent invention, to be used in combination with EGFR pathwayinhibitors to treat preneoplastic/neoplastic diseases associated withabnormal MN expression. Preferred therapies comprise therapies selectedfrom the group consisting of MN-specific antibodies, MN-preferentialcarbonic anhydrase inhibitors, MN antisense nucleic acids, MN RNAinterference, and MN gene therapy vectors; some of which preferredtherapies are described in greater detail below.

Particularly, the EGFR specific inhibitors may be combined with therapyusing MN/CA IX-specific antibodies and/or MN/CA IX-specific antibodyfragments, preferably humanized MN/CA IX-specific antibodies and/orbiologically active fragments thereof, and more preferably fully humanMN/CA IX-specific antibodies and/or fully human MN/CA IX-specificbiologically active antibody fragments. Said MN/CA IX-specificantibodies and biologically active MN/CA IX-specific antibody fragments,preferably humanized and more preferably fully human, may be conjugatedto the EGFR inhibitor, or to a cytotoxic entity, for example, acytotoxic protein, such as ricin A, among many other cytotoxic entities.

MN-Preferential Carbonic Anhydrase Inhibitors

The novel methods of the present invention comprise inhibiting thegrowth of preneoplastic/neoplastic cells with compounds thatpreferentially inhibit the enzymatic activity of MN protein. Saidcompounds are organic or inorganic, preferably organic, more preferablysulfonamides. Still more preferably, said compounds are pyridiniumderivatives of aromatic or heterocyclic sulfonamides. These preferredpyridinium derivatives of sulfonamides are likely to have fewer sideeffects than other compounds in three respects: they are smallmolecules, they are membrane-impermeant, and they are specific potentinhibitors of the enzymatic activity of the tumor-associated MN protein.

The pyridinium derivatives of sulfonamides useful according to thepresent invention can be formed, for example, by creating bonds betweenpyrylium salts and aromatic or heterocyclic sulfonamide reagents, asdescribed in U.S. Patent Application No. 2004/0146955. The aromatic orheterocyclic sulfonamide portion of a pyridinium salt of a sulfonamidecompound can be called the “head,” and the pyridinium portion can becalled the “tail.”

It can be appreciated by those of skill in the art that various otherMN-preferential carbonic anhydrase inhibitors can be useful according tothe present invention.

MN Gene Therapy Vectors

Recent therapeutic strategies proposed to target aggressive tumorsinvolve the utilization of the hypoxia-responsive promoters that candrive the expression of cytotoxic genes selectively in hypoxic tumorcells. This strategy requires that the promoter is turned on in thehypoxic conditions and turned off in the normoxic conditions. Severalapproaches are based on the use of repetitive hypoxia-responsiveelements to achieve a higher magnitude of the hypoxic activation.

MN/CA9 is an excellent candidate for such hypoxia-regulated therapies inthat it is one of the most tightly regulated by hypoxia genes, if notthe most tightly regulated by hypoxia gene, found so far. However, evenMN/CA9 displays some transcription activity under normoxia.

For inhibiting the expression of the MN gene using an oligonucleotide,it is possible to introduce the oligonucleotide into the targeted cellby use of gene therapy. The gene therapy can be performed by using aknown method. For example, either a non-viral transfection, comprisingadministering the oligonucleotide directly by injection, or atransfection using a virus vector can be used, among other methods knownto those of skill in the art. A preferred method for non-viraltransfection comprises administering a phospholipid vesicle such as aliposome that contains the oligonucleotide, as well as a methodcomprising administering the oligonucleotide directly by injection. Apreferred vector used for a transfection is a virus vector, morepreferably a DNA virus vector such as a retrovirus vector, an adenovirusvector, an adeno-associated virus vector and a vaccinia virus vector, ora RNA virus vector.

Materials and Methods

Cell culture: SKRC-01, SKRC-08 and SKRC-17 RCC cell lines were a kindgift from Neil Bander (Weill Medical College, Cornell University, NY).Of these cells, the 01 and the 08 lines overexpressed CA IX proteinwhereas the SKRC-17 cell line did not. The cell lines were regularlymaintained at 37° C. in a 95% air and 5% CO₂ incubator in MinimalEssential Medium (MEM) supplemented with 10% fetal bovine serum (FBS), 2mM glutamine, 2 mM non-essential amino acids (NEAA), 50 IU/ml penicillinand 50 μg/ml streptomycin sulfate and 2.5 μg/ml fungizone. All reagentkits, recombinant proteins, antibodies and other reagents such astrypsin for replating the cells were used according to themanufacturer's recommendations.

EGF dependent phosphorylation of CA IX: SKRC-01 cells, grown to 50%confluency in 60 mm culture dishes, were serum starved by growing themin serum-free medium supplemented with 0.1% FBS overnight. The mediumwas then changed to serum-free medium for a further 2 h. Recombinant EGF(rEGF, Santa Cruz) was dissolved in 10 mM acetic acid containing 0.1%BSA at a stock concentration of 50 μg/ml and increasing amounts of rEGFat final concentrations of 0-50 ng/ml were used to stimulate the serumstarved cells for 30 min. Radioimmunoprecipitation assay buffer (RIPA)used in these studies consisted of 50 mM Tris-HCl pH 7.4, 150 mM NaCl,1.0% NP-40, 0.5% sodium deoxycholate, 0.1% SDS containing the proteaseinhibitor cocktail (Roche Diagnostics) supplemented with 2 mMphenylmethyl sulfonyl fluoride (PMSF) and 1 mM activated sodiumorthovanadate. Total RIPA lysates were prepared and equivalent amountsof RIPA lysates were processed for immunoprecipitation. Briefly, theRIPA lysates that have to be immunoprecipitated were treated with 20 μlof washed 50% suspension of protein A-agarose (Santa Cruz) for 30 min at4° C. to eliminate non-specific protein binding. The beads were removedby centrifuging at 1000 g for 1 min and supernatants were retained. Tothe supernatants, a polyclonal antibody against CA IX (Santa Cruz) wasadded at 1:1000 dilution and subjected to gentle mixing in a rocker at4° C. overnight. The immune complexes were collected by addition of 20μl of 50% suspension of protein A-agarose. The samples were then rockedgently at 4° C. for 1 h. Immunoprecipitates were subjected to gelelectrophoresis and blotted onto PVDF membranes. The blocked membraneswere treated with a monoclonal antibody against phosphotyrosine (PY-20,Santa Cruz Biotechnology, CA) at 1:500 dilution. The membranes werewashed and the blots were finally treated with goat anti-mouseimmunoglobulin (IgG) conjugated to horse radish peroxidase (Santa Cruzat 1:3000 dilution). The signals were revealed with enhancedchemiluminescence. As a negative control, the initialimmunoprecipitation was done by replacing the polyclonal antibody to CAIX with normal rabbit serum (Santa Cruz, 1:500 dilution) and followingthrough the entire procedure. As a control for the amounts of proteinloaded on each SDS-PAGE gel, the PVDF membranes with the transferredimmune complexes from the polyclonal antibody (to CA IX) were probedwith M75 monoclonal antibody to CA IX at a 1:1000 dilution (Bayer Corp,West Haven, Conn.), and the signals were visualized by enhancedchemiluminescence as described earlier. Parallel experiments wereperformed to determine the kinetics of the loss of tyrosinephosphorylation when the same SKRC-01 cells were serum starved andstimulated with 50 ng/ml EGF for 30 min as described earlier. Thestimulus was then removed and the extent of phosphorylation was followedfurther for 90 min.

Preparation of lipid rafts from SKRC-01 cells: Lipid rafts were preparedfrom renal cancer cells according to the method of Goebel with fewmodifications [Goebel et al., Human Immunol, 63: 813-820 (2002)].Briefly, around 4×10⁷ cells were lysed in MES lysis buffer containing 25mM MES (morpholinoethane sulfonic acid), 150 mM NaCl, 0.5% Triton X-100and 2 mM EDTA for 30 min on ice and sonicated very briefly (3 one secondpulses). An equal amount of 85% sucrose made in MES buffered saline(MBS) containing protease inhibitor cocktail at 1× concentration (RocheDiagnostics, Indianapolis, Ind.). Ultracentrifuge tubes were underlayedwith 6 ml of 5% and 6 ml of 35% sucrose in MBS and finally the lysedcell suspension was underlayed with the help of a syringe and needlebelow the 35% sucrose layer. The tubes were spun at 104000 g at 4° C.for 20 h. The lipid rafts located at the interface of 5% and 35% sucroselayers were collected as 1 ml fractions. A 2 μl aliquot of the fractionswas routinely spotted on to nitrocellulose membranes and processed withcholera toxin B-subunit conjugated with horse radish peroxidase (HRP)using the enhanced chemiluminescence method (ECL) to detect rafts.

Co-immunoprecipitation of PI3K with CA IX: Equal aliquots of the RIPAcellular extracts prepared from serum starved SKRC-01 cells and thoseprepared by stimulating the serum starved cells with 20 and 40 ng/ml EGFas described earlier were immunoprecipitated with M75 monoclonalantibody to CA IX (1:500 dilution) and the immune complexes werecollected with Protein A/G Agarose (Santa Cruz). The denatured immunecomplexes were separated on 7.5% SDS-PAGE gels, transferred to PVDFmembranes and blocked and probed with a polyclonal antibody to the p85subunit of PI3K (Lab Vision, Freemont, Calif.). As a negative control,equivalent aliquots of the RIPA extracts used for the above experimentwas separated on another gel and probed for the presence of the p85subunit of PI3K using the same antibody as described above. In somecases, the lipid raft fraction isolated from the SKRC-01 cells that wereserum starved and stimulated with 40 ng/ml EGF was alsoimmunoprecipitated with M75 monoclonal antibody and probed for theco-immunoprecipitating PI3K (p85 subunit).

Phosphorylation status of Akt: To determine the activation of PI3K byinteraction with the tyrosine phosphorylated carbonic anhydrase IXprotein, SKRC-17 cells which do not express CA IX protein weretransiently transfected with vector only (pSG5C) or with wild-type CA IXcloned into pSG5C using the Transfast transfection kit (PromegaCorporation, Madison, Wis.) exactly as described by Zatovicova andcoworkers [Zatovicova et al., J Immunol Meth., 282: 117-134 (2003)]. Thecells that underwent transfection were maintained in the CO₂ incubatorfor 64 h. At that time, the complete medium was replaced with a serumfree medium supplemented with 0.1% FBS to mimic serum starvationconditions and the PI3K inhibitors LY 294002 and wortmannin were addedat the indicated concentrations and the incubation continued for 8 morehours. Before completion of this experiment (i.e., at 72 h), thetransfected cells in the presence or absence of the inhibitors werestimulated for 30 min in the presence of recombinant EGF (50 ng/ml).Whole cell extracts were made with the RIPA buffer and equivalentamounts of the extracts were analyzed on 7.5% denaturing polyacrylamidegels as described earlier. The transferred proteins on the PVDFmembranes were probed with phosphospecific antibodies for Ser 473 or Thr308 of Akt (1:1000 dilution, Akt sampler kit, Cell SignallingTechnologies, Beverly, Mass.). Identical amounts of the extracts wererun on another gel and probed with the antibody to unphosphorylated Akt(1:1000 dilution, Akt sampler kit, Cell Signalling Technologies,Beverly, Mass.) using the same blotting and probing conditions, asdescribed above to verify that equivalent amounts of proteins in eachsample had been analyzed.

Site directed mutagenesis of CA IX and stable transfection studies: Thesingle tyrosine at position 449 of the wild-type CA IX protein waschanged to phenylalanine using the Quick Change XL mutagenesis kit(Stratagene, La Jolla, Calif.) and the mutation (CA IX YF) was confirmedby subsequent sequencing [phosphorylation motif at aa 446-452 mutated toGVSFRPA (SEQ ID NO: 28)]. The “sense” (S) and the antisense (A) primersused for creating this mutation were synthesized from MWG-Biotech AG(Charlotte, N.C.). The S primer was 5′-CAA AGG GGG TGT GAG CTT CCG CCCAGC AGA GGT AG-3′ [SEQ ID NO: 29] and the A primer was 5′-CTA CCT CTGCTG GGC GGA AGC TCA CAC CCC CTT TG-3′ [SEQ ID NO: 30]. SKRC-17 cellsconstitutively expressing either wild-type CA IX or the CA IX YF mutantwere obtained by co-transfection of the recombinant plasmids with themammalian expression vector pcDNA 3.1(neo) (Invitrogen, Carlsbad,Calif.) in a 10:1 ratio using the TransFast transfection kit (PromegaCorp, Madison, Wis.) exactly according to the instructions by themanufacturer. The cells were selected for growth at a G418 concentrationof 600 μg/ml and isolated with the use of cloning cylinders. Thetransfected clones were tested for CA IX expression and expandedfurther. Six individual cell populations were analyzed for CA IXexpression to rule out the effect of clonal variation. As negativecontrols, the same SKRC-17 cells were transfected with empty vectorpSG5C and pcDNA 3.1 and individual clones were selected for G418resistance.

Analysis of HIF-1α in relation to CA IX expression and EGF stimulation:SKRC-01, 08 and 17 cells were serum starved as described earlier andstimulated with 50 ng/ml recombinant EGF. The same experiment was alsoperformed with the SKRC-17 cells stably expressing the empty vector,wild-type CA IX plasmid and the CA IX YF mutant plasmid. RIPA lysateswere prepared from all the cells at the end of each stimulationexperiment. For SKRC-01, 08 and 17 lysates, equivalent proteins wereseparated on denaturing gels, immunoblots were prepared and probed forthe presence of CA IX, Akt, phosho Akt (Ser 473), HIF-1α and HIF-1β. Thepolyclonal antibodies for HIF-Iα and HIF-1β were purchased from NovusBiologicals, Littleton, Colo. The expression levels of total Akt andHIF-1β in these blots also served as a control amount of total proteinseparated on each gel. Immunoblots generated from the EGF stimulated andstably transfected lysates of SKRC-17 cells harboring the negativecontrol, wild-type CA IX and the mutant CA IX were probed for therelative expression of Akt and the phosphorylated Akt (Ser 473) usingthe phospho-Akt pathway sampler kit as described above.

The following examples are for purposes of illustration only and are notmeant to limit the invention in any way.

Example 1 Intracellular Domain of CA IX can be Phosphorylated in an EGFDependent Manner

Since epidermal growth factor receptor (EGFR) signaling is criticallymodulated by its localization in cholesterol rich membranes, and sinceits overexpression is well documented in the poor prognosis of renalcell carcinoma, the inventor investigated the effect of EGFR dependentsignaling on the phosphorylation status of CA IX [Sun et al.,Biochemistry, 41: 6338-6345 (2002); Dancey, J E, J Clin Oncol, 22:2975-2977 (2004)]. The results of these studies are shown in FIG. 1A-C.The CA IX expressing SKRC-01 cells were serum starved and stimulatedwith increasing amounts of recombinant EGF and the RIPA extracts weremade from these stimulated cells. These extracts were used inimmunoprecipitation experiments with a polyclonal antibody to CA IX andthe immune complexes collected were run on a denaturing polyacrylamidegel. The proteins transferred to PVDF membranes were probed for thepresence of phosphotyrosine using a commercially available monoclonalantibody. This resulted in the visualization of the tyrosinephosphorylated version of CA IX as shown in FIG. 1, panel A. As anegative control, the same extracts were immunoprecipitated with acommercially available non-immune rabbit serum, processed the immunecomplexes collected and the resulting blots were probed with the sameanti-phosphotyrosine antibody as described earlier which is shown inFIG. 1, panel B. The presence of equivalent amounts of CA IX used forall the lanes as a loading control are shown in FIG. 1, panel C afterprobing the blots with the M75 monoclonal antibody. These resultsindicate that CA IX is capable of receiving stimulatory signals from theepidermal growth factor receptor and participate in the ensuingsignaling pathways. Since CA IX is also a very stable protein, theinventor investigated how this new CA IX function is regulated. Thekinetics of loss of CA IX tyrosine phosphorylation is shown in FIG. 1Drevealed a complete loss of signal after 75 min post stimulation.

Example 2 Co-Immunoprecipitation of Tyrosine Phosphorylated CA IX andP85 of PI3K

Some of the data from this study have indicated a functional cross-talkbetween CA IX and EGFR signaling pathways and suggests that the tyrosinephosphorylated version of CA IX could participate in the phosphatidylinositol-3 Kinase (PI3K) signaling. To investigate this possibility, theinventor immunoprecipitated the serum starved and EGF stimulatedextracts of SKRC-01 cells with the M75 monoclonal anti-CA IX antibodyand probed the resulting blots for the possible association with PI3K.For this, a polyclonal antibody to the p85 subunit of the PI3K was used,which is shown in FIG. 2, upper panel. This figure shows that in theabsence of any stimulatory signal, under completely serum starvedconditions, there is no association of PI3K with the CA IX protein. Thiswould be expected since the C-terminal Y is not phosphorylated underthese conditions (lane 1). This figure also shows that there is an EGFconcentration dependent increase in the amount of associated PI3K (lanes2 and 3). In some experiments, the association of the tyrosinephosphorylated CA IX with PI3K was also verified in the membrane raftpreparations made from the EGF stimulated SKRC-01 cells (upper panel,lane 4). This shows that CA IX is recruited to the lipid rafts where itcould participate in signal transduction processes. To verify thatequivalent amounts of proteins were loaded in the co-immunoprecipitationexperiments, equivalent amounts of protein extracts were run on anindependent gel and the resulting blot was probed for the presence ofthe p85 subunit of PI3K (lower panel). Based on these observations, theinventor infers that CA IX could be an active participant in the PI3Ksignaling pathways.

Example 3 Activation of Akt by CA IX-pY and PI3K Interaction

The inventor then investigated whether the PI3K activation byassociation with CA IX could be reproduced in a CA IX negative RCC cellline such as SKRC-17 upon transfection with a wild-type CA IX plasmidand to investigate whether this associated PI3K could bepharmacologically blocked. The results of these studies are shown inFIG. 3. SKRC-17 cells were transiently transfected with either thewild-type CA IX containing plasmid or the vector alone and were treatedwith inhibitors in the presence or absence of recombinant EGF. Initialtransfection experiments incorporating negative controls which includedempty vector plasmid (pSG5C) in the absence of stimulating EGF (i.e., CAIX- and EGF-) showed no phosphorylation of either Ser 473 or Thr 308 ofthe Akt enzyme, the target of PI3K. It is well known that the activationof PI3K is triggered by the binding of its SH2 domain containing p85regulatory subunit to phosphorylated tyrosine residues of activatedgrowth factor receptors or their substrates [Vivanco I, and Sawyers C L.Nat Rev Cancer, 2: 489-501 (2002); Vanhaesebroeck et al., Trends BiochemSci, 22: 267-272 (1997)]. Thus, in FIG. 3A in lane 2 from left, there isa significant increase in the Ser 473 phosphorylation of Akt under EGFstimulated conditions as studied by using a phospho-specific antibodyfor this species, upon transient transfection of CA IX, when compared toa relatively decreased phosphorylation level of the same protein in theabsence of transfected CA IX but in the presence of EGF (FIG. 3A, lane1). This implies that the activation of PI3K and subsequentphosphorylation of Ser 473 on Akt by EGF stimulation of CA IX expressingcells could be additive and could act as a synergistic mechanism in theactivation of PI3K. These inferences could have significant implicationswith respect to therapeutic interference. Akt phosphorylation could bepharmacologically reduced by PI3K inhibitors, namely LY294002 andwortmannin (FIG. 3A, lanes 3-6 from left). At the indicatedconcentrations, LY294002 is shown to be a better inhibitor of both thebase level and the CA IX stimulated PI3K activity when thephosphorylation status of Akt Ser 473 is studied. As other negativecontrols, the use of equivalent amounts of the vehicle dimethylsulfoxide(DMSO) that was used to dissolve these inhibitors did not have anyeffect on the PI3K activity (data not shown). As loading controls,equivalent amounts of protein extracts were run on another gel andprobed for the presence of unphosphorylated Akt as a measure of totalAkt, which is shown in FIG. 3B. However, no phosphorylation of threonine308 of Akt was evident in these transfection studies (FIG. 3C).Nevertheless, these studies led the inventor to conclude that theintroduction of the membrane bound CA IX in these CA IX negative RCCcells led to an additive activation of PI3K and subsequent activation ofAkt under conditions of EGF stimulation.

Example 4 CA IX when Stably Transfected, Shows Elevated AktPhosphorylation Under EGF Stimulated Conditions

Since the above studies were done under conditions of transienttransfection, that may or may not reflect physiological conditions, theinventor next investigated whether the phenomenon of Akt phosphorylationcould be seen in SKRC-17 cells (which are CA IX negative) when they aretransfected to express the human CA IX protein in a constitutive manner.For this purpose, the pSG5C-CA IX plasmid was co-transfected withpcDNA3.1-neo plasmid at the same ratio as described by Svastova and G418resistant cells were selected [Zatovicova et al., J Immunol Meth., 282:117-134 (2003); Svastova et al., FEBS Lett., 577: 439-445 (2004)]. Inparallel, SKRC-17 cells stably expressing the CA IX YF mutant proteinwas also selected under identical conditions. As negative controls forthese stable transfection experiments, SKRC-17 cells expressing emptyvectors pSG5C and pcDNA3.1-neo were also selected. The cells were serumstarved and stimulated with 50 ng/ml recombinant EGF as describedearlier. RIPA lysates prepared from these cells were subjected todenaturing gel electrophoresis and immunoblots were probed for theexpression of total and phosphorylated Akt (Ser 473) proteins. Theresults of a typical experiment are shown in FIG. 4A. While the totalAkt amount that was followed in each experimental condition wasequivalent, the differences in the level of Akt phosphorylation was moresignificant in wild-type CA IX expressing SKRC17 cells with higher Aktphosphorylation (FIG. 4A, lane 2) compared to the same cells without CAIX expression, both in the presence of EGF (FIG. 4A, lane 1). Whereas,when the same cells expressed the YF mutant of CA IX, significantly lessphosphorylated Akt (FIG. 4A, lane 4) was detected. The correspondingnegative controls for these CA IX proteins in the absence of EGF (serumstarvation) showed basal levels of Akt phosphorylation (FIG. 4A, lanes 3and 5). These results suggest that among other factors such as EGF/EGFRinduced phosphorylation of Akt, CA IX phosphorylation may be anotherimportant factor contributing to the phosphorylation/activation statusof Akt and that the mutation of this single tyrosine to phenylalanine inthe intracellular domain of CA IX abrogates this Akt activating functionof CA IX. Finally, the inventor investigated whether the extent of Aktphosphorylation can be correlated with the level of CA IX expression incells naturally overexpressing CA IX, and whether the relative increasein the extent of Akt phosphorylation can be translated to an increase inthe expression of HIF-1αlevels in these SKRC cells that inherentlydiffer in their levels of CA IX expression. For these experiments, thesame SKRC-01 and 08 cells which are CA IX positive and SKRC-17 cellswhich are CA IX negative were chosen and the results are shown in FIG.4B. These cells were serum starved and stimulated with 50 ng/mlrecombinant EGF as described earlier, and the relative levels ofexpression of CA IX (panel A), total Akt (panel B), phosphorylated Akt(panel C), HIF-1α (panel D) and HIF-1β (ARNT protein, panel E) werefollowed by immunoblotting with specific antibodies. The resultspresented in FIG. 4B essentially reinforce the concept that in CA IXoverexpressing cells, growth factor stimulation results in a relativeincrease in Akt phosphorylation and an increase in the expression levelof HIF-1α, whereas the expression level of HIF-1β is unchanged. Sinceall these experiments were done under normoxic conditions, these resultswill have important implications for hypoxia dependent and independentmodes HIF-1α expression in the hypoxic core and tumor periphery whereelevated CA IX expression could be seen [Potter and Harris, Brit JCancer, 89: 2-7 (2003)].

DISCUSSION

Even though CA IX expression is widely accepted as a marker of hypoxicregions in tumors, there are increasing number of studies which suggestthat CA IX expression is regulated at multiple levels. Parallel studiesthat have focused on the expression of CA IX and pimonidazole stainingfor hypoxic regions revealed a non-overlapping pattern of expression ofCA IX with hypoxic regions with the CA IX positive areas extendingbeyond regions of hypoxia [Beasley et al., Cancer Res., 61: 5262-5267(2001); Olive et al., Cancer Res., 61: 8924-8929 (2001)]. Varyingamounts of HIF-1α can be detected at mildly hypoxic and even undernormoxic conditions in normal tissues and in cell lines [Richard et al.,J Biol Chem., 275: 26765-26771 (2000)]. CA IX expression was also foundto be regulated by cell density [Kaluz et al., Cancer Res., 62:4469-4477 (2002)]. Its expression is very low in sparse and rapidlyproliferating HeLa cell cultures whereas its synthesis is induced indense cultures, very likely triggered by intermediate oxygen tensions ortransient hypoxia. This process has recently been shown to involve theactivation of the PI3K pathway [Kaluz et al., Cancer Res., 62: 4469-4477(2002)]. Apart from this, CA IX was also expressed in necrotic regionswhich are known to be hypoxic [Leek et al., Brit J Cancer, 79: 991-995(1999)]. But in these necrotic and perinecrotic regions, othermechanisms such as the production of TNF-α, the reactive oxygen species(ROS) and NF-kB plays a role in the production of HIF-1α which in turninduces the expression of its target gene, namely CA IX [Haddad andLand, FEBS Lett., 505: 269-274 (2001)]. This is more so in non-clearcell carcinomas of the kidney such as the papillary type 1 tumors,whereas in clear cell carcinomas with VHL gene inactivation either in aninherited manner or in a sporadic manner, there is a near uniformexpression of CA IX throughout the tumor [Leek et al., Brit J Cancer,79: 991-995 (1999) and Wykoff et al., Cancer Res, 60: 7075-7083 (2000)].Thus, the multiple levels of regulation of expression of CA IX can bevisualized as follows: (1) factors such as frank hypoxia in the core ofthe tumor or VHL gene mutations in clear cell RCC tumors that forceHIF1α stabilization; (2) pericellular hypoxic or mildly hypoxic regionswhich are not hypoxic enough to induce HIF-1α stabilization but induceCA IX at intermediate HIF-1α levels through the participation of thePI3K pathway; (3) regions where necrotic foci are observed where theexpression of HIF-1α can be supplemented by the expression of factorsunique to necrotic foci such as TNF-α, ROS and NF-kB; and (4) regions ofthe tumor which are well supplied by oxygen where the expression ofHIF-Iα can be induced under normoxic conditions through mechanisms suchas the overexpression of several growth factor receptors. Severalclinical studies show a clear relationship between high levels of CA IXexpression in tumors and poor prognosis [Loncaster et al., Cancer Res,61: 6394-6399 (2001); Chia et al., J Clin Oncol, 19: 3660-3668 (2001);and Giatromalonaki et al., Cancer Res, 61: 7992-7998 (2001)].

Clear cell RCCs as well as papillary RCCs exhibit a complex andheterogeneous expression of several growth factors and their receptors,of which the role played by the epidermal growth factor receptor appearsto be very significant [Moch et al., Human Pathol, 28: 1255-1259 (1997);and Uhlman et al., Clin Cancer Res, 1: 913-920 (1995)]. They are almostinvariably characterized by an overexpression of EGFR and the cognateligand TGF-α. Several studies indicated the functional intactness of thestimulatory autocrine loop for this receptor which contributes to cancerdevelopment and progression, including cell proliferation, suppressionof apoptosis, angiogenesis and the metastatic spread [Ramp et al., JUrol, 157: 2345-2350 (1997)]. Several recent studies have shown thatthis EGFR can mediate several signaling pathways on the basis of itsresidence in the cholesterol rich microdomains of the cancer cell[Goebel et al., Human Immunol, 63: 813-820 (2002); and Nanjundan et al.,J Biol Chem, 278: 37413-37418 (2003)]. Modulation of cholesterol levelsin these microdomains has been shown to alter the EGFR function andtrafficking and even contribute to its ligand-independent activation[Chen X., and Resh M. D., J Biol Chem, 277: 49631-49637 (2002)]. Theseobservations suggest that EGFR signaling from its location in the lipidrafts may have significant clinical implications and prompted us to testthe possibility that CA IX could be phosphorylated by this receptor in aligand-dependent manner. The inventor has found that this was indeed soin vitro. Upon ligand binding, the cytoplasmic tail of the EGFR getsautophosphorylated and this process helps in the activation of thetyrosine kinase activity of the receptor. In addition, the P-Tyrresidues in the activated receptor also act as docking sites tocytoplasmic signal transducing adapter molecules that contain the SH2 orthe phosphotyrosine binding (PTB) motifs [Schlessinger J., Cell 103:211-225 (2000); and Yarden, Y. and Sliwkowski, M. Y., Nat Rev Mol CellBiol, 2: 127-137 (2001)]. For the P-Tyr of CA IX, which is not endowedwith any tyrosine kinase (TK) activity it may simply serve as a dockingsite for the same or a different set of signal transducing adaptermolecules. Hence, its localization at the lipid raft regions may offerCA IX with a unique opportunity to recruit and direct a signalingpathway which is similar or different to that orchestrated by the EGFR.Thus, CA IX may play a role in amplifying or diversifying the oncogenicsignaling processes elicited by the EGFR alone in renal cell carcinoma.In this context, knowledge of the complete spectrum of the signaltransducing adapter molecules with which the tyrosine phosphorylated CAIX can interact becomes absolutely essential. This would offer uniqueopportunities to interfere with these signaling processes which may havesignificant therapeutic potential. Inhibition of multiple pathways suchas CA IX phosphorylation, HIF-1α targeted therapies, VEGF-R targetedtherapies and EGFR targeted therapies (as opposed to monotherapy usingthe EGFR antagonists only) would theoretically create an environment inthe RCC cell that closely approximates to a restored pVHL function inclear cell carcinoma, even though in reality, there is a biallelic lossof this tumor suppressor gene or function. Thus, signal transductiontherapeutics that involves several of these pathways will offer newavenues for therapeutic approach for RCC and may possibly synergize withexisting therapies such as those with IL-2 and interferon-α.

The results also implicate the involvement of transmembrane carbonicanhydrase IX in PI3K pathway and suggest that CA IX, PI3K and EGFRsignaling may function in an integrated manner to provide a molecularbasis for the up-regulation of HIF-1α under non-hypoxic conditions inthis cancer. Observations by Kaluz and coworkers [Kaluz et al., CancerRes, 62: 4469-4477 (2002)] previously indicated a requirement for PI3Kactivity for the cell density dependent CA IX expression which mightprovide a link between the cancer-restricted expression of CA IX withthe well established role of the PI3K pathway in tumorigenesis. Theresults reported in this study imply that the expression of CA IX andits signaling through the EGFR pathway would activate the PI3K pathway.This in effect would form the basis for a self-promoting signaling loopwhich might be a poor prognostic factor for clear cell RCC. This wouldalso help in explaining why several tumors that have deregulated PI3Kactivity also have elevated expression of CA IX [Kaluz et al., CancerRes, 62: 4469-4477 (2002)].

Several novel features of Akt activation process need to be highlightedhere. The motif in the intracellular portion of CA IX protein ( . . .GVSYRPA . . . ) [SEQ ID NO: 25], and the consensus motif found bycomparison with the corresponding region in CA XII ( . . . GVXYXPA) [SEQID NO: 26], do not conform to the canonical YXXM motif [SEQ ID NO: 27]preferred by the SH2 domain of class IA PI3K adapter p85 subunit. Thereason for this is still not clear and it certainly warrants furtherstudies. There could be several explanations for this observation whichmight be an exception to the rule. First, since occupation of both SH2domains of the p85 subunit, preferably by two adjacent phosphotyrosinemotifs of the binding protein is necessary for full activation of PI3K,the binding of the GVXYXPA motif to PI3K p85 subunit as seen in thisstudy very likely brings up a relatively weaker activation of the PI3Kenzyme as it may bind to the p85 subunit with a lower affinity[Rordorf-Nikolic et al., J Biol Chem, 270: 3662-3666 (1995)]. Second, itmay also be possible that the GVXYXPA motif in CA IX protein interactswith another signal transducing adapter which in turn interacts with thep85 subunit of PI3K. Third, a non-canonical interaction of the p85subunit with other proteins such as HGF/SCF (hepatocyte growthfactor/scatter factor) receptor and ErbB3-p85 subunit was reportedearlier, which may influence endocytic sorting and internalization[Hellyer et al., Biochem J, 333: 757-763 (1998); Ponzetto et al., MolCell Biol, 13: 4600-4608 (1993); and Wu et al., J Biol Chem, 278:40425-40428 (2003)]. Finally, since CA IX is a very stable protein,unlike many other growth factor receptor proteins or signal transducingadapter proteins that undergo tyrosine phosphorylation, the observationthat CA IX protein undergoes tyrosine phosphorylation in the first placeis unique, and the physiological significance of this observation mayextend well beyond its role in PI3K activation. In this respect, thefull spectrum of all the binding partners of phosphorylated CA IX needsto be characterized.

One of the most important functions of the activated Akt protein is toactivate the mammalian target of rapamycin (mTOR) as shown by numerousstudies [Sekulic A., Cancer Res, 60: 3504-3513 (2000); Kozma S. C., andThomas G., BioEssays, 24: 65-71 (2002); Vogt P. K., Trends Mol Med, 7:482-484 (2001); and Aoki et al., Proc Natl Acad Sci (USA), 98: 136-141(2001)]. The mTOR protein has been shown to be central homeostaticsensor receiving signals from a plethora of agents such as growthfactors, amino acids, nutrients, intracellular ATP levels, oxygenlevels, second messengers to integrate and coordinate the levels ofribosome biogenesis, cell cycle progression and translation initiation.Numerous pharmacological and genetic studies place the PI3K activationprocess upstream of the mTOR pathway [Bjornsti M. A., and Houghton P.J., Nat Rev Cancer, 4: 335-348 (2004); and Abraham R. T., Cell, 111:9-12 (2002)]. Among the many important functions of the activated mTORprotein, the most relevant for these studies is its ability to controlthe cap-dependent translation of certain mRNAs that have unique50-untranslated region secondary structure such as in cyclin D1 andc-myc mRNAs which help in the unrestricted progression from GI to Sphase of the cell cycle. Most notably, the HIF-1α protein is alsosynthesized in this manner [Page et al., J Biol Chem, 277: 48403-48409(2002)]. In most cancers where the PI3K pathway is deregulated, theup-regulated mTOR can contribute to hypoxia independent translation ofHIF-1α [Hudson et al., Mol Cell Biol, 22: 7004-7014 (2002); and Philipset al., J Biol Chem, 280: 22473-22481 (2005)]. But, in the case of renalcell carcinoma, with the loss of function of the VHL gene commonly seenin the clear cell type, there is net accumulation of this hypoxia driventranscription factor due to protein stabilization [Linehan, W. M., andZbar, B., Cancer Cell, 6: 223-228 (2004); and Bjornsti M. A., andHoughton P. J., Nat Rev Cancer, 4: 335-348 (2004)]. This leads to theincreased expression of (apart from CA IX) its growth factor targetgenes such as TGF-α, VEGF and PDGF. These growth factors in turncontribute in activating the mTOR pathway. Thus, in the clear cell RCC,mTOR can be up-regulated both by hypoxia driven as well as hypoxiaindependent pathways and those results place CA IX in the activationprocess of Akt in such a way that it may actually integrate both theseHIF-1α dependent and independent pathways as shown in FIG. 5. Theresults also provide a molecular basis of the positive feed back loopsthat are inherent in such integrated pathways and help in thevisualization of a vicious cycle mediated by CA IX mediated signaling.In particular, the scheme put together in FIG. 5 helps in placing themany functions of VHL protein and its relationship to the CA IX mediatedsignaling in proper perspective. It also represents a working hypothesisfor the significance of over-expression of CA IX in clear cell carcinomaof the kidney. For example: (1) the VHL protein has been shown todown-regulate the expression and transport activity of certain anionexchangers (AE) which are in complex with CA II or the membraneassociated CA IV that facilitates bicarbonate transport [Sterling etal., J Biol Chem, 277: 25239-25246 (2002)]. This suggests that thetransmembrane CA IX could also function in a complex in a similarfashion as other carbonic anhydrases; (2) VHL tumor suppressor proteinis the main regulator for the expression of HIF-1α causing adown-regulation of CA IX expression [Ivanov et al., Proc Natl Acad Sci(USA), 95: 12596-12601 (1998)]; (3) as a component of the hypoxic andnon-hypoxic acidification machinery, CA IX might participate in pHdependent mechanism of nucleolar sequestration of VHL protein [Mekhailet al., Nat Cell Biol, 6: 642-647 (2004)]. Thus, enhanced acidificationof the extracellular environment may produce a feed back loop of adown-regulated VHL environment which might lead to HIF-1α stabilization;(4) pVHL protein has also been shown to be required for efficientblockade of the epidermal growth factor receptor and the autocrine loopsthat are established in RCC [Perera et al., Clin Cancer Res, 6:1518-1523 (2000)]; (5) moreover, expression of wild-type VHL in cellsexpressing a mutated endogenous VHL leads to decreased expression ofTGF-α. TGF-α is a direct target for the VHL tumor suppressor which actsby decreasing the stability of TGF-α mRNA [Knebelmann et al., CancerRes, 58: 226-231 (1998)]. Thus, by facilitating both the EGFR blockadeand targeting the TGF-a mediated autocrine loop, the wild-type VHLprotein can down regulate the vicious cycle of CA IX mediated cellsignaling as put forward in this study; and (6) in addition, wild-typepVHL binds to and inactivates certain atypical protein kinase C familymembers such as PKC zeta and delta [Pal et al., J Biol Chem, 272:27509-27512 (1997)]. In this regard, it is very interesting to note thatsome recent studies have implicated PKC β II as the PDK II kinase thatcan activate Akt at serine-473 [Kawakami et al., J Biol Chem, 279:47720-47725 (2004)]. Thus, it would be logical to expect that pVHL wouldtry to impede the Akt activation process which would in turn activatemTOR pathway as a consequence. Thus, all the phenomena described here goon to characterize the molecular signatures for the progression of clearcell carcinoma of the kidney and obviously, VHL inactivation serves thebest interests of the cancer cell. Placement of CA IX as an activeparticipant in the middle of these signaling pathways as shown by thestudies reported here may further help in the understanding of the roleof VHL and its relationship to the overexpression of CA IX in theseprocesses and justify the therapeutic interference of these pathways.Finally, it is entirely possible that when the enzymatic activity isdown-regulated by the use of specific CA IX inhibitors, the CA IXprotein could still function in its signal transduction capacity. Thiswarrants more investigations that focus on inhibiting CA IX in both itscapacities to arrive at maximum therapeutic benefit. The patient's VHLand PTEN status will also determine the ultimate efficacy of such CA IXtargeted therapies.

ATCC Deposits

The materials listed below were deposited with the American Type CultureCollection (ATCC) now at 10810 University Blvd., Manassus, Va.20110-2209 (USA). The deposits were made under the provisions of theBudapest Treaty on the International Recognition of DepositedMicroorganisms for the Purposes of Patent Procedure and Regulationsthereunder (Budapest Treaty). Maintenance of a viable culture is assuredfor thirty years from the date of deposit. The hybridomas and plasmidswill be made available by the ATCC under the terms of the BudapestTreaty, and subject to an agreement between the Applicants and the ATCCwhich assures unrestricted availability of the deposited hybridomas andplasmids to the public upon the granting of patent from the instantapplication. Availability of the deposits is not to be construed as alicense to practice the invention in contravention of the rights grantedunder the authority of any Government in accordance with its patentlaws.

Deposit Date ATCC # Hybridoma VU-M75 Sep. 17, 1992 HB 11128 MN 12.2.2Jun. 9, 1994 HB 11647 Plasmid A4a Jun. 6, 1995 97199 XE1 Jun. 6, 199597200 XE3 Jun. 6, 1995 97198

Similarly, the hybridoma cell line V/10-VU which produces the V/10monoclonal antibodies was deposited on Feb. 19, 2003 under the BudapestTreaty at the International Depository Authority (IDA) of the BelgianCoordinated Collections of Microorganisms (BCCM) at the Laboratoriumvoor Moleculaire Biologie-Plasmidencollectie (LMBP) at the UniverseitGent, K. L. Ledeganckstraat 35, B-9000 Gent, Belgium [BCCM/LMBP] underthe Accession No. LMBP 6009CB.

The description of the foregoing embodiments of the invention have beenpresented for purposes of illustration and description. They are notintended to be exhaustive or to limit the invention to the precise formdisclosed, and obviously many modifications and variations are possiblein light of the above teachings. The embodiments were chosen anddescribed in order to explain the principles of the invention and itspractical application to enable thereby others skilled in the art toutilize the invention in various embodiments and with variousmodifications as are suited to the particular use contemplated.

All references cited herein are hereby incorporated by reference.

1. A method of treating a mammal for a preneoplastic/neoplastic disease,wherein said disease is characterized by abnormal MN/CA9 geneexpression, comprising administering to said mammal a therapeuticallyeffective amount of a composition comprising an EGFR pathway inhibitorwhich is an anti-EGFR antibody. 2-3. (canceled)
 4. The method of claim1, wherein said anti-EGFR antibody is selected from cetuximab,panitumumab, nimotuzumab, matuzumab, and MDX-447.
 5. The method of claim1, wherein said anti-EGFR antibody is conjugated to an antibody orbiologically active antibody fragment which specifically binds MN/CA IX.6. The method of claim 1, wherein said anti-EGFR antibody is abispecific antibody having a specificity for EGFR and a specificity forMN/CA IX.
 7. The method of claim 1 further comprising administering tosaid mammal radiation and/or a therapeutically effective amount in aphysiologically acceptable formulation of one or more of the followingcompounds selected from the group consisting of: conventional anticancerdrugs, chemotherapeutic agents, different inhibitors of cancer-relatedpathways, bioreductive drugs, gene therapy vectors, CA IX-specificcarbonic anhydrase inhibitors, CA IX-specific antibodies and CAIX-specific antibody fragments that are biologically active, and CA9antisense therapies.
 8. The method of claim 7, wherein said inhibitorsof cancer-related pathways are selected from HIF-1α targeted therapies,VEGF-R targeted therapies, IL-2 and interferon-α, inhibitors of the MAPKpathway, and inhibitors of the PI-3K pathway.
 9. The method of claim 8,wherein said inhibitor of the MAPK pathway is the bis aryl-ureaSorafenib (BAY 43-9006) or an omega-carboxypyridyl substituted urea. 10.The method of claim 7, wherein said gene therapy vectors are targeted tohypoxic tumors.
 11. The method of claim 1, wherein saidpreneoplastic/neoplastic disease characterized by abnormal MN/CA9 geneexpression is selected from the group consisting of mammary, urinarytract, bladder, kidney, urethra, ovarian, uterine, cervical,endometrial, squamous cell, adenosquamous cell, vaginal, vulval,prostate, liver, lung, skin, thyroid, pancreatic, testicular, brain,head and neck, mesodermal, sarcomal, stomach, spleen, gastrointestinal,esophageal, rectal, and colon preneoplastic/neoplastic diseases.
 12. Themethod of claim 11, wherein said preneoplastic/neoplastic diseasecharacterized by abnormal MN/CA9 gene expression is kidney cancer. 13.The method of claim 11, wherein said preneoplastic/neoplastic diseasecharacterized by abnormal MN/CA9 gene expression is renal cellcarcinoma.
 14. The method of claim 1 wherein said disease is a normoxictumor.
 15. The method of claim 1 wherein said disease is a hypoxictumor.
 16. The method of claim 1, wherein said mammal is a human. 17-26.(canceled)
 27. The method of claim 7 wherein said one or more compoundsare selected from the group consisting of CA IX-specific carbonicanhydrase inhibitors, CA IX-specific antibodies and CA IX-specificantibody fragments that are biologically active.
 28. The method of claim27 wherein said one or more compounds are selected from the groupconsisting of CA IX-specific antibodies and CA IX-specific antibodyfragments that are biologically active.
 29. The method of claim 28wherein said compound is a CA IX-specific antibody or a CA IX-specificantibody fragment that is biologically active.
 30. The method of claim29 wherein said CA IX-specific antibody or antibody fragment is eitherhumanized or fully human.
 31. The method of claim 29 wherein said CAIX-specific antibody or antibody fragment is conjugated to a cytotoxicentity.
 32. The method of claim 30 wherein said humanized or fully humanCA IX-specific antibody or antibody fragment is conjugated to acytotoxic entity.
 33. The method of claim 29 wherein said CA IX-specificantibody or antibody fragment is conjugated to an EGFR inhibitor. 34.The method of claim 30 wherein said humanized or fully human CAIX-specific antibody or antibody fragment is conjugated to an EGFRinhibitor.